Overview

  • Product name
    Anti-Diphtheria Toxin antibody [11D9]
    See all Diphtheria Toxin primary antibodies
  • Description
    Mouse monoclonal [11D9] to Diphtheria Toxin
  • Host species
    Mouse
  • Specificity
    ab51882 reacts with Diphtheria toxoid (formaldehyde inactivated Diphtheria toxin).
  • Tested applications
    Suitable for: WB, ELISAmore details
  • Immunogen

    Diphtheria toxoid (formaldehyde inactivated Diphtheria toxin).

  • General notes

    For a recombinant antibody to Diphtheria Toxin, please see - ab209329.

Properties

Applications

Our Abpromise guarantee covers the use of ab51882 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use at an assay dependent dilution. Detects a band of approximately 63 kDa (predicted molecular weight: 58 kDa).
ELISA 1/4000.

Target

  • Relevance
    Corynebacterium diphtheriae is a gram-positive, nonmotile bacteria found in soil and animal feces. C. diphteriae infect the epithelial cells of the upper respiratory tract from where they produce and secrete a potent toxin. This toxin is absorbed and disseminated through lymph channels and blood to the susceptible tissues of the body.
  • Cellular localization
    Secreted
  • Alternative names
    • Corynebacterium diphtheriae toxoid antibody
    • Diphtheria Toxin antibody
    • tox antibody

References

This product has been referenced in:
  • Schmohl JU  et al. Development of a Deimmunized Bispecific Immunotoxin dDT2219 against B-Cell Malignancies. Toxins (Basel) 10:N/A (2018). Read more (PubMed: 29316610) »
See 1 Publication for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer

Thank you very much for your reply.

I am sorry that these suggestions are not more helpful. If you would like to try a different DT antibody please let me know and I can arrange a testing discount for you.

If there is anything else that we can do for you, please don't hesitate to ask and I'll be happy to assist you.

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Question
Answer

Thank you very much for your reply and for sending this additional protocol information and the images.

I've looked through the images and alsodiscussed the situation with some of my colleagues. Based on these images, it looks like the antibody volume was held constant with increasing amounts of protein. All of the mouse antibody ab51882 seems to be binding to the beads, but the protein to antibody ratio needs to be optimized. We would recommend using a minimal amount of protein (1 ug or less) and holding this quantity constant while increasing the volume of the antibody, in order to find the optimal protein:antibody ratio. By using a higher quantity of purified protein, the antibody may be saturated so that it can not bind any more protein.

With the rabbit antibody ab53828 (I mis-typed ab53848 in my previous email), it looks like there may be too much protein loaded onto the gel. I'd suggest a similar protocol with a small amount of protein held constant with increasing amount of antibody. It's possible that the rabbit antibody has a higher binding affinity compared to the mouse antibody, and less of this antibody can be used.

As I mentioned before, we haven't tested any of our DT antibodies in IP so we aren't sure which will be most suitable for this application. If you would like to try one of our other DT antibodies, we do have a testing discount program that you can participate in. I might recommend the goat polyclonal ab19950 instead of one of the other monoclonals, as the polyclonal antibodies will have more binding sites on the protein.

I hope that this will be useful, but please keep me updated of any progress and let me know if you have any questions or if there is anything else that we can do for you, and I'll be happy to help.

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Question

I have a question regarding your Immunoprecipitation protocol and looking for some advice. We are looking to bind CRM197 which also contains Vi antigen, basically we are hoping to bind all of the CRM197 so we can detect the Vi.

What we have done is added 20ug of CRM197 with 1-50uL of MAB AB51882 (MAB to DT) overnight rocking at 4 degrees. The next day 100uL of Protein G Beads were added for 4 hours rocking at 4 degrees.

Then centrifuging at 14,000 rpm for 2 minutes, taking off the supernatant and resuspending the pellet (beads) in the same volume as the supernatant then adding 6x loading buffer (50uL) to both the supernatant and the resuspended pellet, heating for 5 minutes then running on a Coomassie. We are hoping by SDS page Coomassie Stain that all the CRM197 has bound to the beads and is not detected in the supernatant.Also we are only focusing on the binding of CRM197 at this time so there is only CRM197 in the sample

Since we are using CRM197 ,we are using PBS buffer only, although we have tried resuspending the beads in 400uL Lysis buffer.

We are following your protocol with a few exceptions:

Protein G Beads=

100mg Beads to 1mL PBS for 1 hour rocking at RT

Centrifuged at 14,000rpm for 2 minutes

Washed with 1mL PBS, centrifuged

Added 400uL PBS or Lysis buffer (non denaturing Lysis buffer- 20mM Tris-HCL pH 8.0, 137mM NaCl, 10% Glycerol, 2mM EDTA and 1% Triton X-100)

Procedure=

CRM197 + 2-25uL MAB AB51882 ( Also have tried Rabbit to DT AB53828, 2-50uL and mixing of both AB51882 and AB53828, 25uL of each) Overnight at 4 degrees rocking

Added 100uL of Beads x 4 hours at 4 degrees rocking

Centrifuged at 14,000 for 2 minutes

Removed supernatant and resuspended pellet in same volume as supernatant

Added 50uL of 6x Loading Buffer to both supernatant and resuspended pellet, heated at 100 degrees x 5 minutes

Ran on Coomassie

We have also tried binding the antibody to the beads overnight then adding 20ug of CRM197 at 4 degrees for 4 hours then following the procedure above.

Results- Band appears for both the supernatant and the pellet. Depending on the experiment the supernatant band is sometimes greater than the pellet. We are hoping to not to have a band on the Coomassie for the supernatant and a band that matches our control for the resuspended pellet.

Is there another antibody to use that may be better suited for this experiment or if you could give us any advise that may help us bind the CRM197 to the beads, it would be greatly appreciated

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Answer

Thank you very much for contacting us with your questions and for sending the details of your protocol.

I do have a few additional questions just to make sure that I understand the situation:

1) How is the CRM197 prepared prior to the IP? Is it in a denatured conformation?
2) In what buffer is the CRM197 incubated with the antibody? What is the total volume during this incubation (protein, antibody, and buffer)?
3) Is there a reason why the elution was done at 100C for 5 min instead of 50C for 10 min? Have you also tried a double elution?
4) Do you have any images of your results?
5) Have you tried using protein A antibodies with the rabbit polyclonal ab53848 (slightly higher affinity than protein G)?
6) Have the antibodies been tested against the CRM197 protein in a Western blot or ELISA?

We have only tested these antibodies in Western blot and ELISA, so we don't have a specifically optimized IP protocol for them, but I may have some suggestions after looking over the additional details. It is also possible that these antibodies are not suitable for use in IP. Unfortunatley none of our DT antibodies have been tested in IP so we don't know which would be best to use in your assay, however we do have a testing discount program available if you would like to try a differentDT antibody in IP.

I look forward to hearing from you. Please let me know if you have any further questions and I'll be happy to help.

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Question

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.    

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Answer

 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.  

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