With the aim of continuous improvement of our products, Biokit increases the shelf life of bioelisa HIV 1+2 Ag/Ab from 10 to 12 months.
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1 1 NEWS BK NEWS # 377 /MKT DATE : TITLE : SHELF-LIFE EXTENSION OF bioelisa HIV 1+2 Ag/Ab With the aim of continuous improvement of our products, Biokit increases the shelf life of bioelisa HIV 1+2 Ag/Ab from 10 to 12 months. bioelisa HIV 1+2 Ag/Ab is a fourth generation ELISA test for the detection of antibodies to HIV 1, HIV 2 and the HIV-1 p24 antigen. The test design includes HIV-1 gp41 recombinant antigen that gives better specificity and two monoclonal antibodies to HIV-1 p24ag gives better sensitivity. According to the internal and external evaluations, bioelisa HIV 1+2 Ag/Ab assay exceeds the performance of today s state-of-the art HIV assays: Biorad Genscreen HIV1+2 Ultra and Abbott Architect HIV1+2 Combo. The new shelf life will be applied to lots B25834 (480 tests) and B25835 (96 tests) and the future lots produced. The codes and packaging remain the same: bioelisa HIV 1+2 Ag/Ab 96 Tests, Cod bioelisa HIV 1+2 Ag/Ab 480 Tests, Cod As a reminder, attached you will find the main features of bioelisa HIV 1+2 Ag/Ab. We hope this improvement helps you in the promotion and introduction of bioelisa HIV 1+2 Ag/Ab in your market. Yours sincerely
2 2 Ernest Mas Marketing Manager BIO-FLASH Product Manager/Clinical Lab Jordi Gómez OEM Business Manager Joaquín Ortiz Product Manager/Blood Bank Sales Support LatAm Laura Marco BIO-FLASH Product Specialist/Clinical Lab Biokit Marketing Team Ricard Forns Marketing Vice President Montse Armengol Marketing Assistant Sandra Roselló Sales Support Manager Sales Support EEMEAI Joan Vilà Werfen Business Manager Sales Support NOA & WE Carlos Fernández Product Manager Instrumentation Sales Support APAC Veronica Vocero Product Specialist/Blood Bank David Padrosa OEM Account Manager Biokit Phone: Biokit Mktg Fax:
3 bioelisa HIV 1+2 Ag/Ab Product features Recombinant antigens and capture antibody bioelisa HIV 1+2 Ag/Ab uses a combination of two recombinant antigens: gp41 from HIV-1 and gp36 from HIV-2, in addition to a synthetic peptide for HIV-1 subtype O, together with a two monoclonal antibodies against HIV-1 p24 Ag. The new HIV antigens are able to detect HIV type 1 group M-O and HIV type 2 in human serum or plasma. Performance of bioelisa HIV 1+2 Ag/Ab in terms of sensitivity and specificity allowed biokit to achieve the Common Technical Specifications (CTS) requested for CE mark class IIA products. Fourth generation assay The bioelisa HIV 1+2 Ag/Ab is a fourth generation assay for total antibody detection and HIV-1 p24 antigen detection. HIV recombinant antigens and synthetic peptides are present in both solid phase and conjugate. If HIV antibodies (IgG, IgM or IgA) are present in the patient sample, they will bind to the HIV antigens on the plate coating and HIV antigens present in the conjugate. If p24 Antigen is present in the patient sample, it will bind to the capture monoclonal antibody present in the plate coating and capture antibody present in the sample diluent. Biokit, S.A. Barcelona, Spain Can Malé Lliçà d Amunt Tel
4 Components MICROPLATE: 12 strips of 8 breakable wells coated with HIV specific gp36 and gp41 peptides and gp41 protein with two monoclonal antibodies specific to the HIV-1 p24 Ag. NEGATIVE CONTROL Ready-to-use control. It contains human serum negative for HIV antibodies and p24 antigen. POSITIVE HIV-1 CONTROL Ready-to-use control. It contains human serum positive to Anti-HIV 1. POSITIVE HIV-2 CONTROL Ready-to-use control. It contains human serum positive to Anti-HIV 2. POSITIVE HIV p24 CONTROL Ready-to-use control. It contains recombinant HIV-1 p24. CONJUGATE #1: Ready-to-use, blue colour-coded reagent. It contains a mixture of biotinylated antigens in phosphate buffer. CONJUGATE #2 CONC. 100x: Horseradish Peroxidase conjugated with streptavidin in specific stabilizer reagent to be diluted with Conjugate # 2 Diluent. CONJUGATE #2 DILUENT: Ready-to-use orange colour-coded reagent used for the dilution of Conjugate # 2 Concentrate 100x. SAMPLE DILUENT: Ready-to-use red-colour coded reagent. It contains active components and monoclonal p24 antigen antibodies. Used to dilute the sample. SUBSTRATE TMB: Ready-to-use component. It contains Tetra methyl benzidine and Hydrogen peroxide (H2O2) in a citrate buffer. STOP SOLUTION CONC. 25x ready-to-use component. It contains 0,3 M H2SO4 solution. WASH BUFFER CONC. 25x: Saline phosphate buffer. ADHESIVE SEALS: 2
5 Protocol The bioelisa HIV 1+2 Ag/Ab uses in plate sample dilution: 100 µl of sample diluent and 100 µl of sample or control. The 1:2 sample dilution allows spectrophotometric monitoring of sample dispensing at 620 nm. The red colour of the sample diluent changes to a clear red dark colour. Definition of the OD limit must be validated customer by customer. Number of replicates controls have been reduced to allow the maximum well utilization: Negative control by triplicate and rest of Positive control for HIV-1, HIV-2 and p24 Ag, only by singlicate. The sample incubation time is to 60 minutes to reach the required sensitivity of fourth generation HIV assays. Conjugate incubation is split into two separate incubations of 30 minutes each because of the biotin streptavidin system. The substrate incubation is 30 minutes. The total incubation time is 2 hours and 30 minutes. Substrate is ready for use. STEP PROCEDURE Add 100 µl of Sample Diluent. Add 100 µl of sample/control to the diluent and pipette up & down for Sample step mixing. Test Negative control x3 Positive Controls x1. Incubate 60 minutes at 37 C. Wash step Perform wash step 5x. Conjugate #1 Add 200 µl of ready-to-use Conjugate #1 to wells of the microtiter plate. Incubate 30 minutes at 37 C. Wash step Perform wash step 3x. Conjugate #2 Add 200 µl of diluted Conjugate #2 to wells of the microtiter plate. Incubate 30 minutes at 37 C Wash step Perform wash step 5x. TMB Substrate Add 150 µl of TMB Substrate to the wells of the microtiter plate. Colour Development Incubate 30 minutes at C. Stopping Add 100 µl of Stop Solution and read OD at 450nm with reference wavelength at nm in an ELISA microplate reader. 3
6 Performance Sensitivity: Analytical Sensitivity (Limit of Detection HIV-1 p24 Ag) The analytical sensitivity of the test was determined using WHO International Standard HIV-1 p24 Antigen and the BIORAD HIV-1 Ag Standard. The WHO International Standard HIV-1 p24 Antigen and the BIORAD HIV-1 Ag Standard were serially diluted in negative human serum and analysed in duplicate with the test assay. Tables are showing the different dilutions of the HIV-1 Antigen standards, the absorbance results, and ABS/CO values have been prepared, from two different lots. The analytical sensitivity was determined as the concentration of the analyte in the highest dilution of the standards yielding an ABS/CO > 1.0. Lot 1 Lot 2 WHO Int std HIV-1 p24 OD/COV 2 IU/ml IU/ml IU/ml End point 0.32 IU/ml 0.32 IU/ml The sensitivity limit has been estimated at < 0.5 IU/mL Lot 1 Lot 2 Biorad HIV-1 Ag std OD/COV 100 pg/ml pg/ml pg/ml pg/ml pg/ml End point 8.3 pg/ml 8.3 pg/ml The sensitivity limit has been estimated at < 12.5pg/ml The analytical sensitivity for antibody detection was determined using the NIBSC International Standards: British Working Standard HIV-1, British Working Standard HIV-1 1 in 5 dilution and Monitor Sample for HIV-2. Lot 1 Lot 2 OD/COV British Working Std HIV British Working Std HIV-1 diluted 1: Monitor Sample HIV
7 The HIV-1 p24 Ag diagnostic sensitivity was assessed by using the Seracare HIV p24 Antigen Mixed Titer Performance panel PRA204 All samples in this panel are positive for the presence of HIV-1 p24 Ag. In the next graphic it is showed the reactivity in terms of signal OD/COV for the most difficult samples in comparison with other commercial assays. The bioelisa HIV 1+2 Ag/Ab presented the best score in all of them. 5
8 Diagnostic Sensitivity The diagnostic sensitivity of the bioelisa HIV 1+2 Ag/Ab Kit was based on testing characterized HIV-1, HIV-2 and HIV-1 p24 Ag positive samples: - A total of 537 HIV-1 positive samples were evaluated with bioelisa HIV 1+2 Ag/Ab, among them: 28 HIV-1 fresh-same-day collected samples, 85 HIV-1 and 4 HIV-1 Subtype O samples in an European reference lab; 380 HIV -1 samples were evaluated at Biokit and 40 HIV-1 non B subtype samples (and at least 3 samples of each HIV-1 subtype) were evaluated in another European reference lab. bioelisa HIV 1+2 Ag/Ab detected all 537 samples. The sensitivity found was100% (537/537). - A total of 115 HIV-2 positive samples were evaluated with bioelisa HIV 1+2 Ag/Ab, among them: 27 HIV-2 samples in an European reference lab and 88 HIV-2 samples were evaluated at Biokit. bioelisa HIV 1+2 Ag/Ab detected all 115 samples. The sensitivity found was 100% (115/115). - A total of 61 HIV-1 p24 Ag positive samples were evaluated also with bioelisa HIV 1+2 Ag/Ab, among them: 15 samples in an European reference lab, 16 samples were evaluated in another European reference lab and 30 samples at Biokit. bioelisa HIV 1+2 Ag/Ab detected all 61 samples. The sensitivity found was 100% (61/61). - Additional sensitivity studies were carried out by testing a total of 31 seroconversion panels at Biokit and a European reference lab. Results obtained were comparable to the most sensitive commercial method for HIV 1+2 antigen and antibody detection HIV Seroconversion Panels from BBI/Seracare and Zeptometrix were evaluated at Biokit. bioelisa HIV-1+2 Ag/Ab identified correctly all the positive specimens. - In the following table it can be observed the first sample detected as positive in each panel evaluated with bioelisa HIV-1+2 Ag/Ab in comparison with 4th Generation assays (Ag/Ab Assays), 3rd Generation assays (only Ab Assays), assays for HIV p24 antigen and NAT HIV RNA assays. bioelisa HIV-1+2 Ag/Ab showed excellent sensitivity to detect early antigen or antibodies in 16 HIV Seroconversion Panels evaluated. 6
9 bioelisa HIV Ag/Ab HIV p24 Seroconversion HIV Ab Assay NAT HIV Ag/Ab Assay antigen Panel First sample detected positive in the panel BB1-PRB965-n= ZEPT n= ND 4 BBI-PRB954-n= BBI-PRB932-n=9 4 ND ZEPT-9017-n= ZEPT-9014-n= ZEPT-9012-n= ZEPT-9032-n= ZEPT-6243-n= ZEPT-9022-n= ZEPT-9018-n= BBI-PRB939-n=9 6 ND BBI-PRB956-n= BBI-PRB945-n= BBI-PRB955-n= BBI-PRB958-n= Bioelisa HIV-1+2 Ag/Ab detected the first positive sample at the same level as an Elisa for only HIV p24 detection and before than a commercial EIA assay for HIV1+2 Ag/Ab detection In the next graphic it is shown the aggregate score in seven seroconversion panels, in comparison with other commercial assays. The aggregate score means the addition of the first detection day in all panels. A lower aggregate value means earlier detection and higher sensitivity. Maximum number detection days by less sensitive HIV CE mark 3rd generation assay: #238. bioelisa HIV 1+2 Ag/Ab showed the best performance. 7
10 Finally a study was conducted in the Paul Ehrilch Institute in Germany, to assess the sensitivity of bioelisa HIV 1+2 Ag/Ab by comparing reactivity with other commercial assays in 10 seroconversion panels. bioelisa HIV 1+2 Ag/Ab showed the best performance. Maximum number detection days by less sensitive HIV CE mark 3rd generation assay: #471 Specificity The specificity was evaluated by testing a total of 6,465 unselected blood donors' samples at three different sites. - In the first site 3,045 unselected samples including 684 first donors were tested. From this total, 4 samples were reactive. The specificity obtained in this study was 99.87% (3,041/3,045). - In the second site 500 unselected samples from Blood Bank were tested. All 500 samples were negative. The specificity was 100% (500/500) - An internal evaluation of 2,920 unselected samples from Blood Bank was performed. From this evaluation, 6 samples were reactive. The specificity obtained in this study was 99.79%. (2,914/2,920) The final specificity was found to be 6,455/6,465: 99.85% 8
11 Evaluation 1 (3,045 sera) Initial Reactive Repeated Reactive False Positive Specificity % Serum Evaluation 2 (500 sera) Initial Reactive Repeated Reactive False Positive Specificity % Serum Internal Evaluation (1,000 sera + 1,920 plasmas) Initial Reactive Repeated Reactive False Positive Specificity % Plasma Serum Total In addition 200 samples from hospitalized patients have been tested and compared to a reference test. 194 samples were found negative by both tests and six samples were reactive. The six samples were also reactive with other ELISA tests. A specificity of 100% was obtained in this study (194/194). Precision Intra-assay reproducibility - The coefficient of variation obtained for the absorbance values of a HIV-1 positive sample assayed in a minimum of 40 replicates was 3.42%, 5.79% and 4.71% in three lots studied. - The coefficient of variation obtained for the absorbance values of a HIV-2 positive sample assayed in a minimum of 40 replicates was 6.39%, 5.91% and 8.38% in three lots studied. - The coefficient of variation obtained for the absorbance values of a HIV-1 p24 positive sample assayed in a minimum of 40 replicates was 4.08%, 3.44% and 3.71% in three lots studied. Inter-assay reproducibility Four positive samples of different levels were tested in 15 different assays. The coefficients of variation obtained for the ratios absorbance/cut-off of the four samples were 4.87%, 5.37%, 7.49% and 7.54%. Interferences No interference has been found for haemoglobin (500 mg/dl), bilirubin (20 mg/dl), human Albumin (5 g/dl) and triglycerides (500 mg/dl). 9
12 Cross-reactivity To evaluate possible interferences, 125 potential cross-reacting specimens were analyzed. Among those samples, 4 samples (RF+), 4 samples positive for anti-nuclear antibodies (ANA), 5 samples Mononucleosis, 89 samples from other related infectious diseases and 23 samples from pregnant women. No evidence of cross reactivity was found in the samples evaluated. The 2 anti-toxoplasma IgM samples that tested positive were also positive by other HIV assays. Potential interfering samples = 125 RF (Rheumatoid factor) - 4 Elevated IgG - 4 ANA (anti-nuclear Antibodies) - 4 Elevated IgM - 5 Mononucleosis - 5 HSV 1 IgG (Herpes Simplex Virus) - 5 Pregnant women- 23 (including 7 multiparous) HSV 2 IgG (Herpes Simplex Virus) - 4 Syphilis Ab - 6 anti-cmv IgG (Cytomegalovirus) - 5 anti-ebv (Epstein-Barr Virus) - 3 anti-cmv IgM (Cytomegalovirus) - 7 anti-rubella (Rubella Virus) - 4 anti-htlv (Human T-lymphotropic Virus) - 4 anti-vzv IgG (Varicela Zoster Virus) - 2 anti-hcv (Hepatitis C Virus) - 8 anti-vzv IgM (Varicela Zoster Virus) - 2 anti-e.coli (Escherichia coli) - 5 anti-toxoplasma IgG (T. gondii) - 10 HBsAg (Hepatitis B Antigen) - 8 anti-toxoplasma IgM (T. gondii) - 7 Comparison vs. Competitors bioelisa HIV 1+2 Ag/Ab can be compared with the two Premium products on the market: The Biorad Genscreen ULTRA HIV and the Murex HIV Ag/Ab combination. The sensitivity and specificity of bioelisa HIV 1+2 Ag/Ab matches what until now was considered the reference assay in Elisa, the Biorad Genscreen assay. The biokit assay is even better in terms of analytical sensitivity or limit of detection of HIV-1 p24 Antigen. Specificity is also better because of the utilization of an improved gp41 protein. In terms of ease-of-use, the bioelisa HIV 1+2 Ag/Ab incorporates all liquid reagents, in comparison with the Biorad and Murex assays that use lyophilized conjugates. There is a unique disadvantage compared to the Biorad and Murex assay. The bioelisa HIV 1+2 Ag/Ab uses two independent incubations for the conjugate incubation step. This means the total incubation time increases by 30 minutes, but the specificity and background of the assay improves because of this extra wash cycle. 10
13 The internal and external evaluations performed on the bioelisa HIV 1+2 Ag/Ab kit demonstrate better performance with seroconversion panels and the detection limit for p24 Ag. Test name bioelisa HIV 1+2 Ag/Ab Genscreen ULTRA HIV Murex HIV Ag/Ab Ag/Ab Combination Code number G79-01 Method EIA/HRP/TMB EIA/HRP/TMB EIA/HRP/TMB Format 3 - step direct sandwich 2 - step direct sandwich 2 - step direct sandwich Solid phase 12x8 Microtiter wells 12x8 Microtiter wells 12x8 Microtiter wells Tests per kit /480 96/480 Coating Recombinant antigens Synthetic peptide Recombinant antigens Synthetic peptide Recombinant antigens Synthetic peptide HIV-1 gp41 Rec gp160 and Synthetic peptide HIV Group O ENV rec Ag and Synthetic peptide HIV Group O HIV-2 gp36 Synthetic peptide HIV2 ENV rec Ag Anti p24 Ag Two Monoclonal antibody One Monoclonal antibody One Monoclonal antibody Conjugate Conclusion Conjugate #1: Biotinylated HIV Ag Ready to-use Conjugate #2: Streptavidin HRP Concentrate Conjugate #1: Biotinylated polyclonal Ab to p24 Ready to-use Conjugate #2: Antigens labelled with lyophilized Streptavidin HRP A+B 80 µl 25 µl Conjugate #1 75 µl Sample The limit of detection is superior to the most important competitors: Murex-Diasorin and Biorad. Specificity has been also improved by means of the new gp41. The large evaluation with blood donor reported a level of specificity not overcome neither Murex-Diasorin nor Biorad. The two kit presentations: 96 and 480 Test adjust better to the customer needs Conjugate: Antigens and monoclonal antibodies conjugated with lyophilized HRP. Substrate Ready to-use 150 µl Ready to-use Specimen dilution 100 µl Sample diluent 25 µl Sample diluent 100 µl Sample 100 µl Sample Sample incubation 60 min / 37 C 60 min / 37 C 60 min / 37 C Conjugate incubation Conjugate #1: 30 min / 37 C Conjugate #2: 30 min / 37 C Conjugate: 30 min / 37 C Conjugate #2: 30 min / 37 C Substrate incubation 30 min / RT 30 min / RT 30 min / 37 C Cut-off Mean Neg Mean Neg Mean Neg Analytical sensitivity 0.5 IU/ml p24 Ag 0.85 IU/ml p24 Ag 28 pg/ml Biorad std pg/ml Biorad std pg/ml Biorad std Diagnostic Sensitivity 100% 100% 100% Specificity 99.87% 99.72% 99.78% Comments + Best analytical sensitivity + 2h incubation time +Reference assay in the + Best sensitivity - Lyophilized conjugate market for diagnostic + Very low background sensitivity - 2h 30 min incubation time + 2h incubation time - Lyophilized conjugate As per the Biokit internal and external results, the bioelisa 1+2 Ag/Ab has to be considered as a new state-of-the-art assay for HIV detection. 11
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