HRP/Anti-M13 Monoclonal Conjugate
Horseradish Peroxidase conjugated to ontl-M13 Monoclonal
Antibody with Mouse ascites fluid
Product Specification Sheet
for research use only.
Not recommended or intended for diagnosis of disease in humans or animals.
Do not use internally or externally in humans or animals.
Store at-15°C to-30°C.
HRP/anti-M13 Monoclonal Conjugate consists of horse radish Peroxidase conjugated to mouse anti-M13 monoclonal antibody. The antibody purified from BALB/c mouse ascites fluid, reacts specifically with the bacteriophage
M13 major coat protein product of gene Vlll (gp8). The conjugate is supplied in 0.8 ml of 50% glycerol/0.5 X PBS for standard usage at a 1:10000 dilution (200 μl/microplate well). This provides sufficient HRP/anti-M13 Monoclonal Conjugate for 200 microplate (96 well) detection assays using the protocol supplied.
Other materials required
·1 x PBS
To prepare 1 x PBS, add the following to a 1 litre flask.
Distilled water to1 litre. Adjust pH to7.4 with HCL. Sterilization is not necessary.
·1 x Blocking buffer
(2% non-fat dry milk in l x PBS).
Dissolve 20 g of non-fat dry milk in1 litre of 1 x PBS.
Note: Prepare fresh daily
(1 x PBS with 0.05% TweenTM20).
Add 0.5 ml Tween 20 to1 litre of 1 x PBS, mix and use.
Note: Prepare fresh daily
·0.05 M Citric Acid
Dissolve 10.507 g of Citrate monohydrate(mw210.14) in1 litre of distilled water, Adjust the pH to 4.0 with 10 N NaOH (~6 ml of base will be required). Filter sterilize through a 0.22μm filter.
·1 x ABTS Stock Solution
Add 0.22 mg of ABTS to every 1 ml of 0.05 M Citric acid, pH 4.0. Prepare fresh or sterilize and store at 2-8°C until required.
·ABTS Substrate Solution
Immediately before use add 36 μl of a 30% H202 solution to 21 ml of 1 ATBS stock solution for each 96-well microplate.
Safety warnings and precautions
All chemicals should be considered as potentially hazardous We therefore recommend that this product is handled only by tl1lose persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice.
Note that the protocol requires the use of ABTSTM (harmful) and hydrogen peroxide (corrosive). Please refer to the manufacturersi safety data sheets for the safe handling and use of these materials.
Each lot of the HRP/anti-M13 Monoclonal Conjugate is tested by ELISA (enzyme-linked immune-sorbent assay) to verify detection of 2.5 x109 pfu/ml of M13 phage captured by bound anti-M13 Monoclonal Antibody with an A405 >0.6 using ABTS substrate.
NOTE: The ELISA protocol used for the recombinant phage antibody clones should be similar to the protocol used for the original polyclonal or monoclonal antibody from which the new recombinant was derived. The buffer, incubation times/temperatures and antigen concentrations used for the original polyclonal or monoclonal antibody ELISA should be inserted into the following protocol where appropriate.
It is good practice to coat a well for each recombinant phage being assayed with a negative control antigen. Suitable negative control antigens are those that are biochemically similar to the antigen of interest. If the recombinant clone reacts positively with the test antigen and the negative control, then it can be assumed that the recombinant antibody either locks specifity for the test antigen or recognizes a common epitope shared by both antigens. If the recombinant antibody non-specifically binds to many negative controls, then the ELISA protocol should be modified. Refer to the troubleshooting guide for more detail. The reaction of HRP with ABTS [2’2’-azino-bis(3-ethylben-thiazoline- 6-sulphonic acid) diammonium salt; Sigma catalog number A1888] will produce a green colour that is quantitated by reading the absorbance at 405-415 nm (optimum is 410 nm) in a plate reader or spectrophotometer.
Positive clones containing recombinant phage captured by the antigen will produce a visible green colour while structurally similar negative control antigens remain nearly colourless.
ELISA screening of phage recombinant antibodies
1. Dilute the antigen to10 ug/ml in an appropriate buffer; e.g., 1x PBS or 0.05 M Na2CO3(PH9.6).
2. For every recombinant phage clone to be assayed. coat at least one well of a 96-well polyvinyl microplate using 200 ul of the above diluted antigen solution per well. At this time, the negative control antigen can also be applied to o second set of wells.
3. lncubate the microplate for1-2 hours at room temperature in a humidified container (e.g., a sealed container containing moistened paper towels) or suitable incubator.
4. Empty the contents of the microtitre wells. To do this, invert the plate and flick the well contents into a suitable container. If the antigen is a biohazard, pipette or aspirate the well contents into an appropriate biohazard receptable. All solutions that come into contact with a plate coated with a biohazard are assumed to be contaminated.
5. Top the inverted plate on a paper towel to remove any liquid remaining in the wells.
6. Fill each well of the microplate with at least 200 ul of 1 x blocking buffer, and incubate the microplale for1-2 hours at room temperature.
NOTE: At his time. It will be convenient to block wells of an additional microplate if phage will be diluted in microtitre wells instead of in microcentrifuge tubes (see below). Alternatively, phage can be diluted in polypropylene plates.
7. Empty the contents of the microplate and blot it on o paper towel as before.
8. In a separate pre-blocked microplate or in microcentrifuge tubes, dilute the recombinant phage antibody supernatant with an equal volume of 1x blocking buffer and incubate the diluted sample for15-30 minutes at room temperature. This steps necessary to block any non-specific protein-protein interactions that may occur between recombinant antibodies and surrounding proteins. NOTE: Since phage antibodies can stick to unblocked plastic microtitre wells, always add phage antibodies to wells which have been blocked or contain 1 x blocking buffer. Alternatively, polypropylene plates can be used.
9. Add 200 ul of the diluted recombinant phage antibody supernatant to each antigen coated well, and incubate the plate for1-2 hours at room temperature.
10. Empty the contents of the microplate and blot on a paper towel.
11. To wash the wells, immerse the plate in a large container of wash buffer and agitate it to release any bubbles in the wells. Remove the plate from the container invert and flick out the contents. Repeat this process five times.
12. Blot the inverted plate on o paper towel to remove any remaining wash buffer
13. Dilute the HRP/anti-M13 Monoclonal Conjugate1:10 000 in1 x blocking Buffer(1:10000 ,2 µl of HRP/onti-M13 Monoclonal Conjugate to 21 ml of 1 x blocking buffer) for 96-well plate, ～21 ml of diluted HRP/anti-M13 Monoclonal conjugate will be required.
14. Add 200 ul of the diluted HRP/anti-M13 Monoclonal conjugate to each appropriate well of the plate.
15. lncubate the microplate for 1 hour at room temperature.
16. Wash the microplate six times with wash buffer as described above.
17. Prepare the ABTS substrate solution by adding 36 ul of 30% H2O2 to 21 ml of 1x ABTS stock solution for each 96-well plate.
18. Add 200 ul of the ABTS substrate solution prepared as described above to each appropriate well of the plate.
19. ncubate the plate at room temperature for 20-60 minutes, until a suitable green colour has formed.
20. The ABTS-HRP reaction can be read in o microplote reader set at 405-415 nm (optimum is 410 nm). If a microplate reader is unavailable, a spectrophotometer set at a wavelength of 410 nm can be used to quantitate the results. The absorbance reading for the antigen should be at a value of at least 0.2 and at least two to three times higher than the absorbance reading of the negative control.
NOTE: lf recombinant antibodies produce a positive ELISA signal with the test antigen and a variety of negative control antigens, refer to the troubleshooting guide for suggestions.
After one round of panning and reinfection, few or none of the 96 colonies picked showed a positive signal in the ELISA.
A. The microplate rescue of enriched phage clones was not successful. The procedure for rescue should be followed exactly. lt is especially important to spin the plates and remove the supernatant before addition of the final medium in the rescue since any residual glucose may inhibit expression of the recombinant phage antibody.
B. The mouse from which the spleen cells were obtained for mRNA did not mount a successful antibody response. Determine the antigen-specific serum concentration. The titre should be at least1:500 as determined by ELISA or Western blot.
C. An insufficient number of rounds of panning/reinfection were performed. At least two rounds of phage antibody panning/reinfection should be performed to enrich for antigen-specific phage antibodies when hybridoma cells are the starting material and 4-7 rounds when spleen cells from a hyperimmunized mouse are the starting material.
D. The HRP/Anti M13 Monoclonal Conjugate is non-functional. Dilute an M13 phage stock (eg, M13K07) to 5 x109 pfu/ml in l x PBS. Coat on unblocked well of a polyvinal microplate with100 ul of the diluted M13 phage. lncubate for 1 hour at room temperature. Block and wash the microplate as described in the ELLSA procedure and detect bound M13 with the HRP/ anti M13 Monoclonal Conjugate. If the integrity of the conjugate is good, colour development will occur.
E. The antigen-coating concentration, microplates and/or blocking reagents are not optimal for the phage antibodies. If phage antibodies produce a weak positive signal, repeat the ELISA using o higher (up to1 mg/ml)or lower antigen-coating concentration. Use polyvinyl rather than polystyrene microplates and block with 2% non-fat dry milk in PBS rather than with casein or BSA.
F. The phage displayed antibody may be poorly expressed. Concentrate the phage by PEG precipitation for use in the ELISA. It may be helpful to titre the phage to determine the concentration.
Clones show cross-reactivity (high background) with the negative control antigen in the ELISA.
A. Recombinant phage used in the ELISA were PEG-precipiatated. Residual PEG may artificially enhance ELISA signals.
B. Blocking agent (2% non-fat dry milk) was not added to the recombinant phage supernatant. If blocking agent is not added along with the phage, the phage may non-specifically stick to unblocked sites on the surface of the plastic.
C. The negative control antigen may be very similar biochemically to your Antigen, or non-specific hydrophobic protein-protein interactions may be occurring. The recombinant antibody sample can be diluted 1:1 in 2% non-fat dry milk containing 0.2% TritonTM X-l00. Washing the microplates more vigorously between steps may also help. ln addition, a comparison of alternative blocking agents (with and without a detergent such as Tween20) may be necessary to reduce or eliminate cross-reactivity.
The outer wells of the plate give unusually high absorbance readings when compared with the inner wells or unusual patters of high and low absorbance appear on the plate.
Evaporation is occurring in the outer wells. Incubate the plate in a humidified incubator. If unusually high absorbance readings continue to occur in the outer wells, it may be necessary to use only the inner wells of the plate for the assay. Patterns can be the result of static electricity or changes in incubation temperatures between steps. Wipe off the bottom of the microplate with a wet paper towel and equilibrate all reagents, wash buffers, etc. to the required temperature.