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Order Details Antibody code: ab34420 Problem Choose: Problem with standard curve No signal or weak signal High background problem with standard curve and high background Lot number ab34420 (mAb anti-DT) lot.877395 ab51882 (pAb-HRP anti DT) lot. 949834 Purchase order number or preferably Abcam order number we don’t have order number, the antibodies were ordered more than six months ago General Information Antibody storage conditions (temperature/reconstitution etc) 2-8°C in the dark Description of the problem (high background, no signal, non-specific color development, poor standard curve etc.) poor standard curve, no reproducibility of standard curve and high background in sandwich ELISA setup. Sandwich ELISA Sample (Species/Cell type/Cell line etc.) Diphtheria Toxoids Coating well (Buffer/Concentration of the coating material etc.) Carbonate buffer 50mM, pH 9.6 Blocking conditions (Buffer/time period, Blocking agent etc.) 2h 37°C, Milk 5% in PBST 1X Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) ab34420 (mAb anti-DT) diluted in carbonate buffer 50mM, pH 9.6 2. at 1ug/mL, 1H 37°C, washed three times with 100ul/well PBST Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) ab51882 (pAb-HRP anti DT) diluted 1:1000 in Milk 2.5% PBST, 1H 37°C, washed three times with 100ul/well PBST Detection method (Substrate/Diluent etc.) TMB Sigma 100uL/well after 15min added H2SO4 1N 100 ul/well Positive and negative controls used (please specify) Diphtheria Toxoids 12 Lf/mL (positive control) PBST 1X (Negative control) Optimization attempts (problem solving) How many times have you tried the ELISA? more than 10 times to set up the assau Have you run a "No Primary" control? yes Yes No Do you obtain the same results every time? Yes No no What steps have you altered? IAb dilution, to set up the assay Additional Notes: we tested ab34420 (mAb anti-DT) lot.877395 in indirect ELISA, poor or no standard curve detected varying the mAb concentration. Data: below and exemplum of the results we obtained during sandwich ELISA set up. Coating 1ug/ml in carbonate buffer 50mM, pH9.6 ab34420, Blocking 1h 37°C in PBST and Milk 5%, Standard curve from 48.5 Lf/mL (serial dilution 1:2), IAb-HRP diluted 1:1000 and 1:500 in PBST and Milk 2,5%, development 15min, TMB. std. Curve Lf/mL ab51882 dil 1_1000 ab51882 dil 1_500 48,5 0,377 0,384 0,518 0,492 24,25 0,263 0,282 0,318 0,397 12,125 0,279 0,273 0,319 0,316 6,0625 0,175 0,206 0,299 0,317 3,03125 0,145 0,187 0,155 0,189 1,515625 0,112 0,14 0,426 0,156 0,7578125 0,093 0,16 0,188 0,151 0,37890625 0,074 0,078 0,142 0,13 0,189453125 0,077 0,079 0,124 0,138 0,094726563 0,085 0,077 0,144 0,058 0,047363281 0,086 0,109 0,199 0,099 Bkg 0,072 0,094 0,141 0,143 We would appreciate if you are able to provide a copy of the data, particularly from the standard curve. This will help us to assess the results.
Asked on Nov 22 2011
Thank you for your reply and for taking the time to complete our questionnaire. The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality. Reviewing this case, I would like to offer some suggestions to help optimize the results from ab51882 and ab34420. I would also appreciate if you can confirm some further details: 1. I can suggest to try coating the plates overnight at 4oC. This can sometimes improve the coating. 2. The concentration of antibodies may require increasing for optimization of results. Have higher concentrations been tried? 3, Could you confirm if the substrate solutions have been prepared immediately before use and are in date? 4. As discussed in the previous email, I am sorry these antibodies are regrettably not tested and guaranteed in sandwich ELISA (sELISA). It may be they are not suitable for use together in sELISA. These antibodies have been tested in indirect ELISA, and we can provide our 6 month guarantee for this application. Could you provide the results from the indirect ELISA's? I can suggest to try the same suggestions for indirect ELISA to help optimize the results. In the event that a product is not functioning in the tested applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund. I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.
Answered on Nov 22 2011