HIV (1+2) Ag&Ab
Contents
1. as to achieve optimal performance during testing 2 Make sure that all reagents are within the validity indicated on the kit box and are of the same lot Never use reagents beyond the expiry date stated on reagents labels or on the kit box 3 CAUTION CRITICAL STEP Allow the reagents and samples to stabilize at room temperature 18 30 C before use Shake reagent gently before and return to 2 8 C immediately after use 4 Use only sufficient volume of sample as indicated in the procedure steps Failure to do so may cause in low sensitivity of the assay 5 Do not touch the bottom exterior of the wells Fingerprints or scratches may interfere with microwell reading 6 When reading the results ensure that the plate bottom is dry and there are no air bubbles inside the wells 7 Never allow the microplate wells to dry after the washing step Immediately proceed to the next step Avoid the formation of air bubbles when adding the reagents 8 Avoid assay steps long time interruptions Assure same working conditions for all wells 9 Calibrate the pipette frequently to assure the accuracy of samples reagents dispensing Always use different disposal pipette tips for each specimen and reagents as to avoid cross contaminations Never pipette solutions by mouth The use of automatic pipettes is recommended 10 Assure that the incubation temperature is 37 C inside the incubator 11 When adding samples avoid touching the we2. of possible deterioration of the reagents and or operator or equipment errors In such case the results should be considered as invalid and the samples must be retested In case of constant erroneous results classified as due to deterioration or instability of the reagents immediately substitute the reagents with new ones 2 If after mixing of the Chromogen A and B solutions into the wells the color of the mixture turns blue within few minutes do not continue carrying out the testing and replace the reagents with fresh ones VALIDITY Please do not use this kit beyond the expiration indicated on the kit box and reagent labels REFERENCES 1 Barbe F et al 1994 Early detection of anti bodies to HIV 1 by a third generation enzyme immunoassay Ann Biol Clin Paris 52 341 345 Barre Sinoussi F et al 1984 Isolation of a T lymphotropic retrovirus from a patient at risk for acquired immunodeficiency syndrome AIDS Science 220 868 871 Clave F et al 1991 Solution conformation preferences of immunogenic peptides derived from the principal neutralization determination of the HIV 1 envelope glycoprotein gp120 Biochemistry 30 9187 9194 Constantine N T et al 1993 Serologic test for the retroviruses approaching a decade of evolution AIDS 7 1 13Gnann JW et al 1987 Science 237 1346 1349 Barbe F et al 1994 Early dtection of antibodies to HIV 1 by a third geneartion enzyme immunoassay Ann Bi
3. sandwich complex comprising of the coated antibodies p24 biotinylated antibodies The microwells are then washed to remove unbound serum proteins The detection of the captured HIV p24 antigen biotinylated antibody complex or HIV 1 2 antibodies is achieved during the second incubation step by adding of the enzyme Horseradish Peroxidase HRP which has been conjugated to second HIV 1 2 recombinant antigens and to avidin p24 detection When p24 has been captured inside the wells avidin will react with the biotin and attach HRP to the Ab p24 Ab complex HIV 1 2 antibody detection When HIV 1 2 antibodies have been captured inside the wells the HRP conjugated antigens will bind to the captured antibodies forming Ag Ab Ag HRP sandwich immunocomplex The microwells are washed to remove unbound conjugate and Chromogen solutions are added to the wells In wells containing the Ag Ab Ag HRP and or Ab p24 Ab HRP sandwich immunocomplexes the colorless Chromogens are hydrolyzed by the bound HRP to a blue colored product The blue color turns yellow after stopping the reaction with sulfuric acid The amount of color intensity can be measured and is proportional to the amount of antibodies or p24 captured in the wells and to the sample respectively Wells containing samples negative for anti HIV 1 2 or p24 remain colorless COMPONENTS 96 Tests MICROWELL PLATE 1 plate Blank microwell strips fixed on white st
4. the skin or eyes ProClin 300 used as a preservative can cause sensation of the skin The enzymatic activity of the HRP conjugates might be affected from dust reactive chemical and substances like sodium hypochlorite acids alkalis etc Do not perform the assay in the presence of such substances Materials Safety Data Sheet MSDS available upon request If using fully automated microplate processing system during incubation do not cover the plates with the plate cover The tapping out of the remainders inside the plate after washing can also be omitted ASSAY PROCEDURE Step 1 Reagents preparation Allow the reagents and Step 2 Step 3 Step 4 Step 5 Step 6 samples to reach room temperature 18 30 C for at least 15 30 minutes Check the Wash buffer concentrate for the presence of salt crystals If crystals have formed resolubilize by warming at 37 C until crystals dissolve Dilute the Wash Buffer 1 to 20 with distilled or deionized water Use only clean vessels to dilute the buffer Numbering Wells Set the strips needed in strip holder and number sufficient number of wells including three Negative controls e g B1 C1 D1 three Positive controls one for HIV 1 one HIV 2 and one for HIV Ag controls e g E1 F1 G1 and one Blank e g A1 neither samples nor HRP Conjugate should be added into the Blank well Use only number of strips required If the results will be determined by using dual wavelength plat
5. For Research Use Only MpressBio HIV 1 2 Ag amp Ab Catalog WI4496 Not for Diagnostic Use HUMAN IMMUNODEFICIENCY VIRUSES ANTIGEN ANTIBODY ELISA KIT Two Step Incubation Double Ag amp Ab Sandwich Principle INSTRUCTIONS FOR USE This Screening HIV 1 2 Ag amp Ab ELISA is an enzyme linked immunosorbent assay ELISA for qualitative determination of antigens or antibodies to Human Immunodeficiency Virus HIV type 1 and or type 2 in human serum or plasma For Research Use Only SUMMARY The human immunodeficiency viruses type 1 and type 2 are the etiological agents of the acquired immunodeficiency syndrome AIDS and related conditions HIV has been isolated from patients with AIDS AIDS related complex ARC and from healthy individuals at high risk for AIDS Infection with HIV is followed by an acute flu like illness This phase may remain unnoticed and the relationship to HIV infection may not be clear in many cases The acute phase is typically followed by an asymptomatic carrier state which progresses to clinical AIDS in about 50 of infected individuals within 10 years after seroconversion Serological evidence of infection with HIV may be obtained by testing for presence of HIV antigens or antibodies in serum of individuals suspected for HIV infection Antigens can generally be detected during both acute phase and the symptomatic phase of AIDS only The Antibodies to HIV 1 and or HIV 2 can be detected throughout virtual
6. alyzed 1 The OD value of the Blank well which contains only Chromogens and Stop solution is less than 0 080 at 450 nm 2 The OD value of the Positive control must be equal to or greater than 0 800 at 450 630 nm or at 450 nm after blanking 3 The OD value of the Negative control must be less than 0 100 at 450 630 nm or at 450 nm after blanking 3 Interpretations of the results S the individual absorbance OD of each specimen Negative Results S C O lt 1 Samples giving absorbance less than the Cut off value are negative for this assay which indicates that no HIV 1 2 antibodies or p24 antigen have been detected with this Screening HIV 1 2 Ag amp Ab ELISA kit Positive Results S C O 2 1 Samples giving an absorbance equal to or greater than the Cut off value are considered initially reactive which indicates that HIV 1 2 antibodies and or p24 antigen have probably been detected using this Screening HIV 1 2 Ag amp Ab ELISA kit Retesting in duplicates of any initially reactive sample is recommended Repeatedly reactive samples could be considered positive Borderline S C O 0 9 1 1 Samples with absorbance to Cut off ratio between 0 9 and 1 1 are considered borderline and retesting of these samples in duplicates is recommended to confirm the results LIMITATIONS 1 Non repeatable positive result may occur due to the general biological characteristics of the ELISA method The assay is designed to achieve v
7. d or contaminated and sufficient volume of Wash buffer is dispensed each time into the wells 5 In case of manual washing we suggest to carry out 5 cycles dispensing 350 400 ul well and aspirating the liquid for 5 times If poor results high background are observed increase the washing cycles or soaking time per well 6 In any case the liquid aspirated out the strips should be treated with a sodium hypochlorite solution at a final concentration of 2 5 for 24 hours before liquids are wasted in an appropriate way The concentrated Washing solution should be diluted 1 to 20 before use For one plate mix 50 ml of the concentrate with 950 ml of water for a final volume of 1000 ml diluted Wash Buffer If less than a whole plate STORAGE AND STABILITY The components of the kit will remain stable through the expiration date indicated on the label and package when stored between 2 8 C do not freeze To assure maximum performance of this Screening HIV 1 2 Ag amp Ab ELISA kit during storage protect the reagents from contamination with microorganism or chemicals PRECAUTIONS AND SAFETY This kit is intended FOR RESEARCH USE ONLY The ELISA assay is a time and temperature sensitive method To avoid incorrect result strictly follow the test procedure steps and do not modify them 1 Do not exchange reagents from different lots or use reagents from other commercially available kits The components of the kit are precisely matched
8. d solution 2 0M H2804 1 vial 1 vial PLASTIC SEALABLE BAG 1 unit For enclosing the strips not in use CARDBOARD PLATE COVER 2 sheets To cover the plates during incubation and prevent evaporation or contamination of the wells PACKAGE INSERTS 1 copy ADDITIONAL MATERIALS AND INSTRUMENTS REQUIRED BUT NOT PROVIDED 1 Freshly distilled or deionized water 2 Disposable gloves and timer 3 Appropriate waste containers for potentially contaminated materials 4 Disposable V shaped troughs 5 Dispensing system and or pipette single or multichannel disposable pipette tips 6 Absorbent tissue or clean towel 7 Dry incubator or water bath 37 1 C 8 Microshaker for dissolving and mixing conjugate with samples 9 Microwell plate reader single wavelength 450 nm or dual wavelength 450 nm and 630 nm 10 Microwell aspiration wash system SPECIMEN COLLECTION TRANSPORTATION AND STORAGE 1 Sample Collection Either fresh serum or plasma samples can be used for this assay Blood collected by venipuncture should be allowed to clot naturally and completely the serum plasma must be separated from the clot as early as possible as to avoid hemolysis of the RBC Care should be taken to ensure that the serum samples are clear and not contaminated by microorganisms Any visible particulate matters in the sample should be removed by centrifugation at 3000 RPM for at least 20 minutes at room temperature or by fil
9. e reader the requirement for use of Blank well could be omitted Where appropriate the requirement for use HIV 2 positive control could be omitted Adding Biotin conjugated reagent Add 20ul of biotinylated anti HIV p24 antibodies into each well except in the Blank Adding Samples Add 100ul of Positive controls Negative controls and Specimen into their respective wells Note to avoid cross contamination use a separate disposable pipette tip for each specimen Negative or Positive Control Incubating 1 Cover the plate with the plate cover and incubate for 60 minutes at 37 C It is recommended to use thermostat controlled water tank to assure the temperature stability and humidity during the incubation If dry incubator is used do not open the door frequently Washing 1 At the end of the incubation remove and discard the plate cover Wash each Step 7 Step 8 Step 9 Step 10 Step 11 well 5 times with diluted Wash buffer Each time allow the microwells to soak for 30 60 seconds After the final washing cycle turn the plate down onto blotting paper or clean towel and tap it as to remove any remaining liquids Adding HRP Conjugate Add 100ul HRP Conjugates into each well except in the Blank Incubating 2 Cover the plate with the plate cover and incubate for 30 minutes at 37 C Washing 2 After the end of the incubation remove and discard the plate cover Wash each well 5 times with diluted Wash b
10. eparately when calculating and interpreting the results regardless of the number of plates concurrently processed The results are calculated by relating each sample s optical density OD value to the Cut off value C O of the plate If the Cut off reading is based on single filter plate reader the results should be calculated by subtracting the Blank well OD value from the print report values of samples and controls In case the reading is based on dual filter plate reader do not subtract the Blank well OD from the print report values of samples and controls 1 Calculation of the Cut off value C O NC 0 12 NC the mean absorbance value for three negative controls Example 1 Calculation of NC Well No B1 C1 D1 Negative Controls OD value 0 032 0 031 0 027 NC 0 030 2 Calculation of Cut off C O 0 030 0 12 0 150 If one of the Negative Control values does not meet the Quality control range specifications it should be discarded and the mean value calculating again using the remaining two values If more than one negative control OD value does not meet the Quality control range specifications the test is invalid and must be repeated 2 Quality control range The test results are valid if the Quality Control criteria are verified It is recommended that each laboratory must establish appropriate quality control system with quality control material similar to or identical with the patient sample being an
11. ery high performance characteristics of sensitivity and specificity and the sandwich model minimizes the unspecific reactions due to interference with unknown matters in sample Antibodies or p24 may be undetectable during the early stages of the disease and in some immunosuppressed individuals 2 Common sources for mistakes are kits beyond the expiry date bad washing procedures contaminated reagents incorrect assay procedure steps insufficient aspiration during washing failure to add samples or reagents equipment timing volumes sample nature and quality 3 The prevalence of the marker will affect the assay s predictive values 4 If after retesting of the initially reactive samples the assay results are negative these samples should be considered as non repeatable false positive and interpreted as negative As with many very sensitive ELISA assays false positive results can occur due to the several reasons most of which are related but not limited to inadequate washing step 5 The assay cannot distinguish between infections with HIV 1 and HIV 2 6 The assay cannot distinguish between positive antibody and positive p24 antigen results 7 This is a qualitative assay and the results cannot be use to measure antibodies concentrations INDICATIONS OF INSTABILITY OR DETERIORATION OF THE REAGENTS 1 Values of the Positive or Negative controls which are out of the indicated Quality control range are indicator
12. icantly increases the assay s sensitivity comparing to the previous generations In addition the detection of IgM antibodies that are present only during the early stages of infection much shortens the antibody detection window period the period of time in which there is no detectable antibody production and compare to the second generation sandwich tests could detect antibodies 11 days earlier To reduce even further the antibody detection window period 4 generation HIV ELISAs that could simultaneously detect HIV antigens p24 and antibodies have been developed and are commercially available since 1998 With detection of p24 the 4 generation tests shorten the window period to 16 days or compare to the 3 generation HIV infection could be detected 8 days earlier PRINCIPLE OF THE ASSAY This Screening HIV 1 2 Ag amp Ab ELISA is a two step incubation sandwich enzyme immunoassay kit which uses polystyrene microwell strips pre coated with recombinant HIV antigens recombinant HIV 1 gp41 gp120 and recombinant HIV 2 gp36 and anti HIV p24 antibodies As a first step biotinylated anti HIV p24 antibodies together with the patients serum or plasma sample are added into the wells During incubation the specific HIV 1 2 antibodies if present in sample will be captured inside the wells Simultaneously if HIV p24 antigen is present in sample it will also be captured as a double antibody
13. ll s bottom with the pipette tip 12 When reading the results with a plate reader it is recommended to determine the absorbance at 450 nm or at 450 nm with reference at 630 nm 13 All specimens from human origin should be considered as potentially infectious 14 Materials from human origin may have been used in the kit These materials have been tested with tests kits with accepted performance and found negative for antibodies to HIV 1 2 HCV TP and HBsAg However there is no analytical method that can assure that infectious agents in the specimens or reagents are completely absent Therefore handle reagents and specimens with extreme caution as if capable of transmitting infectious diseases Strict adherence to GLP Good Laboratory Practice regulations can ensure the personal safety Never eat drink smoke or apply cosmetics in the assay laboratory 15 Bovine derived sera may have been used in this kit Bovine serum albumin BSA and fetal calf sera FCS are derived from animals from BSE TSE free geographical areas 16 17 18 19 20 The pipette tips vials strips and sample containers should be collected and autoclaved for 1hour at 121 C or treated with 10 sodium hypochlorite for 30 minutes to decontaminate before any further steps for disposal The Stop solution 2M H2SO 4 is a strong acid Corrosive Use it with appropriate care Wipe up spills immediately or wash with water if come into contact with
14. ly the whole infection period starting at or shortly after the acute phase and lasting till the end stage of AIDS The 1 generation tests were based on viral lysate antigens derived from viruses that are grown in human T lymphocyte lines The presence of traces of host cell components in which the virions have been propagated could lead to cross contamination and thus to very high rates of false positive results With the cloning of the HIV genome improved assays based on recombinant proteins and or synthetic peptides known as 2nd generation became rapidly available The utilization of biotechnology methods allow predominantly expression of the important immunoreactive regions of the proteins and also enabled the production of combined HIV 1 HIV 2 assays The recombinant antigen could also be produced with considerably more purity and in large amounts and they can be bond to solid phase surface with much tighter control over protein ratios and concentrations The first and second generations HIV kits were based on indirect ELISA method and could detect IgG antibodies only by enzyme labeled anti human IgG antibody The third generation ELISA utilized double antigen sandwich method again with antigens coated on solid phase polystyrene plates but with antibodies detection achieved with the help of another enzyme labeled antigen The third generation assays could detect all antibodies in sample IgG IgM etc which signif
15. ol Clin Paris 52 341 345 Barr P J et al 1987 Antigenicity of domains of the HIV envelope polypeptide expressed in the yeast Saccharomyces cerevisiae Vaccine 5 90 101 Express Biotech International P O BOX 458 Thurmont MD 21788 USA Tel 301 228 2444 Fax 301 560 6570 Toll Free 888 562 8914 www xpressbio com info xpressbio com XpressBio Life Science Products
16. rip holder The plate is sealed in aluminum pouch with desiccant 8 12 12x8 well strips wells per plate Each well contains recombinant HIV 1 2 antigens and anti p24 antibodies The microwell strips can be broken to be used separately Place unused wells or strips in the plastic sealable storage bag together with the desiccant and return to 2 8 C NEGATIVE CONTROL Yellowish liquid filled in a vial with green screw cap 1 ml per vial Protein stabilized buffer tested non reactive for HIV 1 2 Preservatives 0 1 ProClin 300 Ready to use as supplied Once open stable for one month at 2 8 C ANTIBODY POSITIVE CONTROL 1 HIV 1 Red colored liquid filled in a vial with red screw cap 1 ml per vial Antibodies to HIV 1 diluted in protein stabilized buffer Preservatives 0 1 ProClin 300 Ready to use as supplied Once open stable for one month at 2 8 C ANTIBODY POSITIVE CONTROL 2 HIV 2 Red colored liquid filled in a vial with yellow screw cap 1 ml per vial Antibodies to HIV 2 diluted in protein stabilized buffer Preservatives 0 1 ProClin 300 Ready to use as supplied Once open stable for one month at 2 8 C ANTIGEN POSITIVE CONTROL Red colored liquid filled in a vial with blue screw cap 1 ml per vial HIV p24 recombinant antigen diluted in protein stabilized buffer Preservatives 0 1 ProClin 300 Ready to use as supplied Once open stable for one month at 2 8 C HRP CONJUGATE REAGENTS 1 vial Red colored liq
17. tration on 0 22 um filters Plasma samples collected into EDTA sodium citrate or heparin may be tested but highly lipaemic icteric or hemolized samples should not be used as they could give erroneous results in the assay Do not heat inactivate samples This can cause sample deterioration 2 Transportation and Storage Store samples at 2 8 C Samples not required for assaying within 3 days should be stored frozen 20 C or lower Multiple freeze thaw cycles should be avoided For shipment samples should be packaged and labeled in accordance with the existing local and international regulations for transport of clinical samples and ethological agents SPECIAL INSTRUCTIONS FOR WASHING 1 A good washing procedure is essential to obtain correct and precise analytical data 2 It is therefore recommended to use a good quality ELISA microplate washer maintained at the best level of washing performances In general no less than 5 automatic washing cycles of 350 400 ul well are sufficient to avoid false positive reactions and high background 3 To avoid cross contaminations of the plate with sample or HRP conjugates after incubation do not discard the content of the wells but allow the plate washer to aspirate it automatically 4 Anyway we recommend calibrating the washing system on the kit itself in order to match the declared analytical performances Assure that the microplate washer liquid dispensing channels are not blocke
18. uffer as in Step 5 Coloring Dispense 50ul of Chromogen A and 50ul Chromogen B solution into each well including the Blank cover the plate with plate cover and mix by tapping the plate gently Incubate the plate at 37 C for 30 minutes avoiding light The enzymatic reaction between the Chromogen solutions and the HRP produces blue color in positive control and HIV 1 2 positive for antigens antibodies sample wells Stopping Reaction Remove and discard the plate cover Using a multichannel pipette or manually add 50ul Stop Solution into each well and mix gently Intensive yellow color develops in Positive control and HIV 1 2 positive for antigens antibodies sample wells This Screening HIV 1 2 Ag amp Ab ELISA TOTAL 297 0 297 EIA 1 HIV Ag Ab 0 203 203 TOTAL 297 203 500 AGREEMENT 297 203 500 100 TOTAL 2 1 3 EIA 3 HIV Ab 0 2682 2682 TOTAL 2 2683 2685 AGREEMENT 2 2682 2685 99 96 TOTAL 0 1 1 EIA 4 HIV Ag Ab 2 2682 2684 TOTAL 2 2683 2685 AGREEMENT 0 2682 2685 99 89 Step 12 Measuring the Absorbance Calibrate the plate reader with the Blank well and read the absorbance at 450 nm If a dual filter instrument is used set the reference wavelength at 630 nm Calculate the Cut off value and evaluate the results Note read the absorbance within 10 minutes after stopping the reaction INTERPRETATION OF RESULTS AND QUALITY CONTROL Each microplate should be considered s
19. uid filled in a white vial with red screw cap 12 ml per vial Horseradish peroxidase conjugated HIV 1 2 antigens Horseradish peroxidase conjugated avidin Ready to use as supplied Once open stable for one month at 2 8 C BIOTIN CONJUGATE REAGENT Blue colored liquid filled in a vial with blue screw cap 3 ml per vial Biotinylated anti HIV p24 antibodies diluted in protein stabilized buffer Preservatives 0 1 ProClin 300 Ready to use as supplied Once open stable for one month at 2 8 C 1 vial 1 vial 1 vial 1 vial 1 vial STOCK WASH BUFFER 1 bottle Colorless liquid filled in a clear bottle with white screw cap 50 ml per bottle pH 7 4 20 x PBS Containing Tween 20 as a detergent DILUTE BEFORE USE The concentrate must be diluted 1 to 20 with distilled deionized water before use Once diluted stable for one week at room temperature or for two weeks at 2 8 C CHROMOGEN SOLUTION A Colorless liquid filled in a white vial with green screw cap 6 ml per vial Urea peroxide solution Ready to use as supplied Once open stable for one month at 2 8 C CHROMOGEN SOLUTION B Colorless liquid filled in a black vial with black screw cap 6 ml per vial TMB solution Tetramethyl benzidine dissolved in citric acid Ready to use as supplied Once open stable for one month at 2 8 C STOP SOLUTION 1 vial Colorless liquid filled in a white vial with yellow screw cap 6 ml per vial Diluted sulfuric aci
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