Enzyme Linked Immunosorbent Assay (ELISA) Kit

(Cat #:  mELISA-25AP/mELISA-50AP, rELISA-25AP/rELISA-50AP)

Product Description Sheet

       Product:  Enzyme Linked Immunosorbent Assay (ELISA) kit

      Catalog #:   mELISA-25AP and mELISA-50AP (Contains sufficient reagents to process 25 or 50 96        well ELISA plates)

      Kit Contents:                                                                                mELISA-25AP/mELISA-100AP

               Peptide/protein binding buffer(5X) Cat. # FGI-1960                       150/300 ml

               Wash buffer      (5X) Cat. # FGI 1961                                              250/500 ml

               High potency blocking buffer (5X) Cat. # FGI-1962                         150/300 ml

               Dry Block Powder (Fat Free Dry milk powder with additives)          40gm/80gm

               Antibody Dilution buffer (5X) Cat. # FGI 1963                                  150/300 ml

               Secondary Antibody AP Conjugate (goat anti-mouse IgG, A, M)       100/200 ul

               Alkaline Phosphatase substrate buffer (5X)       Cat. # FGI-1964      50/100 ml

               Alkaline Phosphatase substrate tablets.  Cat. # FGI-1965        50/100 tablets

               Stopping Buffer (10X).  Cat. # FGI-1966                                          25/50ml ml

       Reagents/Instruments provided by investigator:

                  ELISA plates

                  Antigenic peptides/protein or competing antigenic protein

                  ELISA reader

                  Automatic pipettes or pipetting station

                  Humidified chambers at 25-40^oC

                  Orbital shaker.

(The contents of this kit must be stored at 4^oC)

       Introduction

In order to test the effectiveness of immunization, the pre-immune serum and the test bleeds are monitored for the presence of antibodies against a given antigen.  The test is carried out using Enzyme Linked Immmuno-Sorbent Assay (ELISA).  The test is based on double antibody sandwich assay, the antigen (peptide, protein, others) is immobilized in wells of polystyrene plates.  After blocking non-specific binding sites, the antibodies were allowed to bind to the immobilized antigen.  The excess antibodies are removed by sequential washing and a enzyme (alkaline phosphatase or horseradish peroxidase) linked secondary antibody is allowed to bind to the first antibodies.  The excess of secondary antibodies removed and the presence of secondary antibody is measured by measuring enzymatic activity associated with secondary conjugates in the presence of color producing substrate solution.  After optimum color development the enzymatic reaction is stopped by addition of stop solution and the optical density is measured in spectrophotometer. 

       Procedure:

 In order to get consistent and reproducible results using ELISA-25 or ELISA-100 kit, the  following steps are performed. 

I.                    Preparation of ELISA plates          

1.      Clean high binding capacity flat bottom 96 well ELISA plates are numbered and prepared for antigen tagging.  Note:  Prepare several plates for each antigen, the antigen tagged plates can be stored and used at a later date for titer determination on subsequent bleeds. 

2.       Prepare the antigen solution at 10ng/ml in peptide/protein binding buffer.  Dilute stock peptide/protein binding buffer with 4 volumes of water and serially dilute the antigen to 10ng.ml concentration.

3.      Pippet 50 ul of peptide/protein solution (10ng/ml) to all the wells except the last well (well # H-12).  This will serve as reagent blank.  Alternatively, the antigen solution can be added directly in the desired wells as determined by ELISA reader program format. 

4.      The plates are covered with lids, sealed with Parafilm^TM  and kept in humidified chambers at 25^oC for overnight incubation. 

5.      Remove antigen solution and rinse all wells with 200 ul of wash buffer.  Decant and tap plates on a thick stack of filter papers to remove residual solution.  Note:  The wells should be dried at this time to increase the sensitivity of the assay and reduced background.

6.      Prepare 25 ml blocking solution by mixing 1 gm of dry blocking powder with 5 ml of 5X blocking buffer.  Add 250 ul of blocking buffer to each well, cover and incubate for 2 hours.  Discard the remaining solution.

7.      Aspirate the blocking buffer, wash plate with 250 ul of wash buffer for 5 minutes with shaking.  Remove wash buffer and repeat wash step one more time.  The dry plates can be stored at 4oC under vacuum sealed plastic pouches. 

                           II.   Primary Antibody incubation:

             1.      In a new 96 well ELISA plate, serially dilute test serum in antibody dilution buffer                    at 1:10, 1:100, 1:1000., 1:10K and 1:100K by taking 22 ul test serum in 200 ul
                   of antibody.  Mix and transfer 22ul of solution to the next well containing 200 ul                    antibody solution. 

2.      Serially dilute the pre-immune or normal serum. 

3.      Transfer 50 ul of the serially diluted antibodies to respective pre-tagged antigen plates.

4.      Incubate sealed plates in humidified chambers at room temperature for 120 minutes with shaking. 

5.      Aspirate the antibody and tap on thick layer of filter papers to remove residual antibody form the wells

6.      Wash each well with 250 ul of wash buffer for 10 minutes with shaking.  Remove and discard all solution, tap dry the wells.  Repeat wash steps 3 more times. Note:  Do not allow the plates to go dry, keep the plates after washing in humidified chambers before moving to the next step. 

III.               Secondary Antibody incubation:

1.      Freshly prepare 5 ml/plate of secondary antibody (anti-mouse or anti-rabbit IgG, A, M) in antibody dilution buffer.  Dilute 2 ul of antibody solution in 50 ml of antibody dilution buffer.   .

2.      Pipette 50 ul of secondary antibody in all wells and incubate in humidified chamber at room temperature for 1 hour.   

3.      Aspirate secondary antibody and tap dry ELISA plate.  Wash all wells with 250 ul of wash buffer.  Incubate for 10 minutes with shaking. Aspirate and tap-dry the plate.  Repeat wash step 3 more times.

IV.       Color development and quantitation.

1.      Prepare substrate solution by dissolving two 5mg p-nitrophenyl phosphate (PNPP) in 10 ml of 1X substrate solution.  Note:  Substrate buffer concentration can be reduced 10-fold depending upon the buffering capacity desired.   We recommend that optimization for this step is necessary only if there is high back- ground or false positive are obtained.  Avoid touching the substrate tablets with hands. 

2.       Add 100 ul of substrate solution to each well and incubate at room temperature for 30 minutes or until sufficient color is developed. 

3.      Add 50 ul of stop solution prepared by diluting 1 part of stock with 9 parts of distilled water.  The plates can be stored at 4oC for up to several hours with out any appreciable change in color density.

4.    Read the absorbance at 405 nm, Alternatively, the absorbance can be monitored in a kinetic ELISA reader. Antibody titer is expressed as reciprocal of the serum dilution that results in twice the optical density obtained by pre-immune or normal serum at similar dilution.  If the ELISA titer is below 10,000, we do monitor subsequent bleeds, however, if the titer does not come up by bleed 5, we recommend to change the carrier protein, conjugation chemistry or try using a different adjuvant in new host species. 

V.        General Recommendations

We recommend the optimization of these reagents for you application may be necessary only if the standard protocol gives high background or false positive results.  If high background is consistently obtained, the following steps are recommended:

                Avoid touching or scratching the wells with pipette tips at all time. Use clean plastic and glassware to avoid contamination. 

                Optimize the washing steps by increasing the number of washes, wash buffer volume and incubation time.

                Increase the dilution of secondary antibody to 1:10,000

                Reduce the concentration of substrate buffer.  

                Reduce the incubation time with substrate solution.

                Include a true positive control (by incubating 10 ul of secondary antibody solution with 100 ul of substrate solution), Negative control (by omitting the secondary antibody) and a reagent blank (by omitting the primary antibody).

Storage:

All contents of the kit are stable for 4-6 months at 4⁰C.  Freeze secondary antibody at -20oC if not to be used for more than a month.  Do not freeze/thaw.  Dissolve by warming the solution at room temperature any precipitate that may form after long-term storage at 4⁰C.

* For users who may require more than 5 of mELISA or rELISA-25 or 50 kits,  please inquire about bulk material discounts from FabGennix International Inc..

This Product is for Research Use Only and is NOT intended for use in humans or clinical diagnosis.                                                                                    071003-0010SF1001Z-rev10.00