Helena Laboratories

1 The Helena TITAN GEL Serum Protein System is intend-ed for the separation and quantitation of serum proteinsby agarose gel contains over one hundred individual proteins,each with a specific set of functions and subject to spe-cific variation in concentration under different the introduction of moving-boundary electrophore-sis by Tiselius2and the subsequent use of zone elec-trophoresis, serum proteins have been fractionated onthe basis of their electrical charge at a particular pH intofive classical fractions: albumin, alpha1, alpha2, beta andgamma proteins. Each of these classical electrophoreticzones normally contains two or more fifteen serum proteins have been studiedextensively because they may be measured are large molecules composed of covalentlylinked amino acids. Depending on electron distributionsresulting from covalent or ionic bonding or structural sub-groups, proteins can be either polar or nonpolar at agiven pH.

2 In the TITAN GEL Serum Protein procedure,proteins are separated according to their respective elec-trical charges at on agarose gel using both theelectrophoretic and electroendosmotic forces present inthe system. The proteins are then stained with AmidoBlack staining TITAN GEL Serum Protein GelIngredients:Each gel contains agarose in barbitalbuffer with thimerosal added as a : FOR IN-VITRO DIAGNOSTIC for Use:The gels are ready for use and Stability:The gels should be stored atroom temperature (15 to 30 C) and are stable untilthe expiration date indicated on the package. Thegels must be stored in the protective packaging inwhich they are shipped. DO NOT REFRIGERATEOR FREEZE THE of Deterioration:Any of the following condi-tions may indicate deterioration of the gel: (1) crys-talline appearance indicating the agarose has beenfrozen, (2) cracking and peeling indicating drying ofthe agarose, (3) bacterial growth indicating TITAN GEL Serum Protein BufferIngredients:The buffer is a barbital-sodium barbitalbuffer with sodium azide added as a preserva-tive; pH , TexasHelenaLaboratoriesPro 513/00(7)WARNING: FOR IN-VITRO DIAGNOSTIC USEONLY.

3 DO NOT buffer contains barbital which, in sufficient quan-tity, can be toxic. To prevent the formation of toxicvapors, sodium azide should not be mixed with discarding reagents containing sodi-um azide, always flush sink with copious quantities ofwater. This will prevent the formation of metallicazides which, when highly concentrated in metal, arepotentially explosive. In addition to purging with water,plumbing should occasionally be decontaminatedwith 10% for Use:Dissolve one bag in 1500 mL ofdeionized water. The buffer is ready for use when allmaterial is completely and Stability:The packaged buffer shouldbe stored at 15 to 30 C and is stable until the expira-tion date indicated on the package. Diluted buffer isstable two months at 15 to 30 of Deterioration:Discard packaged buffer ifthe material shows signs of dampness or discol-oration.

4 Discard diluted buffer if it becomes Amido Black Protein StainIngredients:When reconstituted as directed, thestain contains (w/v) Amido Black : FOR IN VITRO DIAGNOSTIC USEONLY. DO NOT for Use:Dissolve the dry stain (entirecontents of vial) in 1 L of the Fixative/Destain Solutionmade in the Materials needed but not provided sec-tion. Mix thoroughly for 30 and Stability:The dry stain should bestored at 15 to 30 C and is stable until the expirationdate indicated on the package. The diluted stain isstable one year stored at 15 to 30 of Deterioration:The diluted stain should be ahomogeneous mixture free of precipitate. Discard ifprecipitate high quality scanning densitometer with visibletransmittance capability may be used to scan the is the Helena EDC (Cat.)

5 No. 1376), theCliniScanTM2 (Cat. No. 1260) or the CliniScan 3 ( 1680). Refer to the Operator s Manual for COLLECTION AND HANDLINGS pecimen:The specimen may be serum, plasma, urineor cerebrospinal fluid. Use of plasma will cause a fibrino-gen band to appear as a distinct narrow band betweenthe beta and gamma Factors:1. Hemolysis may cause false elevation in the alpha2and beta of age, while the other fractions have reachedadult levels by this time. Adult levels of the gamma glob-ulins are not reached until 10-16 years of age. The albu-min decreases and beta globulin increases after the ageof Testing RequiredThe serum protein electropherogram or densitometrictracing should be evaluated for abnormalities. If abnor-malities are observed, appropriate follow-up studiesshould be initiated.

6 These may include immunoelec-trophoresis, immunofixation, quantitation of immunoglob-ulins, bone marrow examination and other OF RESULTS5, 6 Results on normal individuals will cover age and sex-related variations and day-to-day biologic states in which abnormal patterns are observedinclude inflammatory response, rheumatic disease, liverdiseases, protein-loss disorders, monoclonal gam-mopathies, pregnancy and genetic all electrophoretic procedures are nonlinear, it iscritical to use the recommended volume of undilutedserum to obtain optimal resolution and reproducibleresults. Noncompliance with the recommended proce-dure may affect the PERFORMANCE CHARACTERISTICSP recision:Within-Run and Run-to-Run precision studiesyielded CV s of less than 10%.Sensitivity:The sensitivity of the system, using theAmido Black Protein Stain, is 10 :A comparison study of this method to thecellulose acetate method, using a range of g/dL, was excellent yielding a linear regressionequation of Y = + (where X is the TITANGEL method and Y is the cellulose acetate method) anda correlation coefficient of Alper, , Plasma Protein Measurements as aDiagnostic Aid, N Eng J Med, 291:287, Tiselius, A.

7 , A New Approach for ElectrophoreticAnalysis of Colloidal Mixtures, Trans Faraday Soc,33:524, Ritzmann, and Daniels, , DiagnosticProteinology:SeparationandChar acterizationofProteins,QualitativeandQua ntitativeAssays,inLaboratoryMedicine, Harper and Row, Inc.,Hagerstown, Ritzmann, and Daniels, , SerumProteinAbnormalities:DiagnosticandC linicalAspects, AllenLess Co., Killingsworth, et al., Protein Analysis, Diag Med,3-15, Jan/Feb, Killingsworth, , Plasma Protein Patterns in Healthand Disease, CRC Crit Rev in Clin Lab Sci, August, Tietz, , ed., TextbookofClinicalChemistry, 3rded., Saunders Co., Philadelphia, pg 524, GEL SERUM PROTEIN KIT Cat. No. 3041 TITAN GEL Serum Protein Gels (10)TITAN GEL Serum Protein Buffer (1 pkg.)Amido Black Protein Stain (1 vial)TITAN GEL Blotter A (20)TITAN GEL SPE Templates (10)Other Supplies and EquipmentThe following items, needed for performance of theTITAN GEL Serum Protein Procedure, must beordered Microdispenser and Tubes6210 TITAN GEL (Incubation and Drying Oven)5116 TITAN GEL Multi-Staining Set1558 SPE Control (1 x 2 mL)5136 Titan Plus Power Supply1504 Titan Blotter Pads5037 For Sales, Technical and Order Information and ServiceAssistance, call 800-231-5663 toll Laboratories warrants its products to meet our published specifications and to be freefrom defects in materials and workmanship.

8 Helena s liability under this contract or otherwiseshall be limited to replacement or refund of any amount not to exceed the purchase price attrib-utable to the goods as to which such claim is made. These alternatives shall be buyer s exclu-sive no case will Helena Laboratories be liable for consequential damages even if Helena hasbeen advised as to the possibility of such foregoing warranties are in lieu of all warranties expressed or implied including, but not lim-ited to, the implied warranties of merchantability and fitness for a particular GEL Serum Protein SystemCat. No. 3041 Shaded areas indicates that text has been modified, added Inaccurate results may be obtained on specimens leftuncovered, due to and Stability:Fresh serum or plasma is thespecimen of choice. If storage is necessary, samplesmay be stored covered at 15 to 30 C for 4 days or 2 to6 C for 2 weeks, or -20 C for 6 and urine specimens may be used after proper con-centration (10-50X) with a provided:The following materials needed forthe procedure are contained in the TITAN GEL SerumProtein Kit (Cat.)

9 No. 3041). Individual items are not GEL Serum Protein Gels (10)TITAN GEL Serum Protein Buffer (1 pkg)Amido Black Protein Stain (1 vial)TITAN GEL Blotter A (20)TITAN GEL SPE Templates (10)Materials provided by Helena Laboratories but notcontained in the kit:ITEMCAT. Microdispenser and Tubes6210 SPE Control5136 TITAN GEL (Incubator, Oven, Dryer)5116 Titan Plus Power Supply1504 TITAN GEL Multi-Staining Set1558 Titan Blotter Pads5037 Materials needed but not provided:Glacial Acetic AcidMethanolFixative/Destain Solution: Mix 1 L methanol, 1 Ldeionized water and 200 mL glacial acetic acid. Mixwell. Use 1 L of this solution to prepare the stain solu-tion and the remainder for destaining the OF CONDITIONS Gel .. TITAN GEL Serum Protein Gel Buffer Dilution .. 1500 mLSample Dilution .. 1 part sample + 3 parts bufferSample Volume.

10 3 LSerum Absorption Time.. 4 minutesElectrophoresis Time .. 15 minutesVoltage .. 120 VDrying Time .. 5 minutesStaining Time .. 10 minutesDestaining Time .. 2 x 1 minuteDrying time (after destaining) .. 5 minutesScanning Wavelength .. 595 nmRecommended EWC Parameters:Buffer volume; mL per chamber section .. 20 mLElectrophoresis Voltage .. 85 VElectrophoresis Time .. 20 minutesStaining Time .. 10 minutesIncubation Time/Temp .. N/ADrying Time .. 15 minutesDrying Temperature .. 55 CSTEP-BY-STEP METHODA. Preparation of the TITAN GEL CHAMBER1. Dissolve one bag of TITANGEL Serum Protein Bufferin 1500 mL of Pour approximately 25 mLof diluted buffer into eachinnersectionof the Cover the chamber until ready to Sample Application1. Dilute each patient sample and control 1:4 (1 partsample + 3 parts buffer) with TITAN GEL SerumProtein Remove the TITAN GEL Serum Protein Gel fromthe protective packaging.