2. Preparation  and  Determination  of  Optimal


Dilutions  of Enzyme  Conjugates


I.Anti-Human-IgG Penicillinase Conjugate :


A. Principle :


Glutaraldehyde is an extensively used cross linking agent for the conjugation of penicillinase with other proteins. It reacts irreversibly with e-amino group of lysine residues of enzyme and the antibody resulting in the formation of a crosslinkage. Single step method is widely accepted for the conjugation of penicillinase to antibody.


B.Materials :


a) Penicillinase enzyme


b) Anti-human IgG


c) 0.25 M Sodium phosphate buffer (SPB) , pH 7.2 (Appendix-I)


d) 0.05 M Sodium phosphate buffer (SPB) , pH 7.2 (Appendix-I)


e) 0.01 M Sodium phosphate buffer (SPB) , pH 7.2 (Appendix-I)


f) 0.01 M Phosphate buffer saline containing 0.05 % Tween-20 (PBS/T)  (Appendix-I)


g) 3 % Gelatin


Dissolve 3 g of gelatin in 100 ml of 0.05 M SPB (pH 7.2) and heat indirectly until it is dissolved completely.


h) 1 % Glutaraldehyde


    Add 0.1 ml of glutaraldehyde (25%)  to 2.4 ml of distilled water.


i) Penicillinase substrate


   (Starch-iodine-penicillin 'V' reagent)    (Appendix-I)


j) Pooled positive and negative sera.


k) M.tb ES-31/41 antigen cocktail  (Appendix-II) .




C.Methods :


Preparation of conjugate (Single step glutaraldehyde method) :


1.Take 0.5 ml of anti-human IgG (approximately 5 mg protein)  in  a  plastic tube, add 1000   units of enzyme penicillinase, make upto 2.0 ml with SPB (0.25 M, pH 7.2)  and mix.


2.Add 20 to 30 µl of 1% glutaraldehyde while stirring the mixture and keep at 250C for 2 hr with intermittent shaking.


3. Take the mixture in a dialysis bag and dialyse at  40 C against SPB (0.01 M, pH  7.2) for12  hr. with 3 buffer changes (each time 1 litre) .


4. Centrifuge and add sodium azide (0.01%) to the supernatant and store at 40C.Determination of optimal dilution :


1.Apply 5 µl of optimally diluted M.tb ES-31 antigen in 0.05M SPB, pH 7.2 ( 200 ng protein/ml)  on CAM square of plastic stick.


2.Allow to dry at room temperature for 20 to 30 min.


3.Incubate the sticks in 3% gelatin solution in a small beaker at 370C for 2 hr.


4.Make 1:600 dilution of pooled positive serum and pooled negative serum by diluting 10 µl serum to 6 ml in PBS/T.


5.Wash the sticks 3 times with PBS/T (For washing add PBS/T, shake gently and  discard after 1 minute. Repeat this 2 times) .


6.Add 0.5 ml of diluted pooled positive serum into each of 10 small  plastic vials. Similarly add 0.5 ml  diluted pooled  negative  serum into  each of another set of 10 plastic vials.


7.Transfer one antigen coated (gelatin blocked) CAM stick to each tube and incubate at 370C for 1 hr.


8.Wash sticks 5 times with PBS/T. (Discard PBS/T after 3 minutes during each wash) .


9.Prepare serial (two fold)  dilutions of anti human IgG- Penicillinase conjugate in PBS/T. ( starting from  1: 500 to 1: 8000)  and add 0.5 ml of each dilution into one positive serum and one negative serum incubated tubes. Incubate at 370C for 30 min.


10.Wash 9 times with PBS/T.


11.Change sticks to labelled glass tubes.


12.Add 0.5 ml of starch-iodine-penicillin 'V' substrate into each tube, incubate at 370C and evaluate the results visually.


13.Note  the time  of  decolorization  of  substrate  in the  tubes. Optimal  dilution of the conjugate  is  the highest  dilution  showing decolorization of substrate with positive serum about 4 minutes earlier than with negative serum.






Some immunoglobulin preparations give heavy precipitate after glutaraldehyde is added.   To prevent this, first add only 6 µl of glutaraldehyde followed by 2 µl each time at intervals of 1 minute. Stop the addition if slight precipitate formation is observed.


The substrate is blue in color due to the formation of starch-iodine complex. The enzyme penicillinase converts penicillin 'V' to penicilloic acid, which in turn removes iodine from   starch-iodine complex making the substrate colorless.


II.Preparation of Bm mf ES Ag-peroxidase conjugate by periodate method :


A.Principle :


The periodate method was introduced by Nakane and Kawaoi in 1974 and modified by Wilson and Nakane. This gives excellent enzyme antibody (IgG)  ratios. It involves the oxidation of peroxidase with m-periodate and generates some proportion of conjugate in polymeric form. However, this does not usually present a problem, and the conjugation procedure is uncomplicated and gives reliable results. For this reason it is a recommended procedure.




a.Sodium carbonate bicarbonate buffer-1 pH 9.5 (Appendix-I)


b.Sodium periodate 0.2 M (4.3 mg in 1 ml D.D.W.)


c.Sodium acetate 1.0 mM pH 4.4      (Appendix-I)


d.Sodium carbonate bicarbonate buffer-2, pH 9.5 (0.2M) (Appendix-I)


e.Borate buffer, 0.1M pH 7.4  (Appendix-I)


f.Sodium borohydrite solution (4 mg in 1 ml of D.D W)


g.Bm mf ES antigen


h.HRPO [Sigma]


C. Method :


1.Dialyse Bm mf ES Ag overnight against sodium carbonate bicarbonate buffer 1 pH 9.5.


2.Dissolve 2 mg of HRPO in 0.5 ml D.D.W.


3.Prepare  fresh 0.2 M solution  of  sodium m-Periodate  (NaIO4)   & then add 0.1ml


to HRPO solution in a glass bottle-the mixture immediately turns green.


4.Stir the bottle gently for 20 min on shaker at Room Tempature (250C)


5.Dialyse against 1.0 mM sodium acetate buffer pH 4.4 at 40C for 5 hrs with 4-5 changes


6.Raise  the pH  9.0-9.5  by addition  of  approximately 10 ml of 0.2M sodium carbonate bicarbonate buffer 2 pH 9.5. Immediately add 500 µg of Bm mf ES Ag.


7.Stir the mixture gently on shaker for 2 hrs at room temperature (250 C) .


8.Add 100 ml of freshly prepared sodium borohydride solution. Keep the mixture for 2 hr at 40C.


9.Dialyse the conjugate  overnight  against 0.1 M borate buffer pH 7.4 at 40C with 2-3 changes.


10.Add BSA to the conjugate to a final  conc. of  5 mg/ml, distribute into aliquates in vials and  add  one granule of  thymol & store at -200C.




Determination of optimal dilution :


1.Add and incubate  16 wells in a  microtitre plate with Rabbit anti BM mf 'S'  antibodies (5ng/100 ml in corbonate buffer, pH 9.6)  and equal number of wells with  only carbonate buffer (control wells)  at 370 C for 3 hrs.


2.Wash once with PBS/T (for washing add PBS/T in each well shake gently and  discard after 1 minute) .


3.Remove the contents and add 100 µl of 1 % BSA in 0.06M Carbonate buffer pH 9.6 and incubate at 370C for 2 hrs.


4.Wash twice with PBS/T (as described above) .


5.Add 100 ml of  two fold  serial dilutions (1:1,000 to 1:16,000)  of BM mf ES antigen -HRPO   conjugate  diluted in  PBS / T  ( each dilution  to  be added in dupicate in both antibody coated and control wells )  and incubate  at 370C.


6.Wash 5 times with PBS/T (as described above) .


7.Add 100 ml OPD substrate  and  incubate for  10 min. at room temperature (25OC)  in dark.


8.Stop  the reaction  by  adding 50  ml  of  5N  HCI to each well and  read  the OD using ELISA reader.


9.The conjugate in highest dilution which gives  maximum difference between antibody coated and control wells  can be considered as optimal dilution.


Note :


  The absorbance of control well should be < 0.1 and that of the antibody coated well should be > 1


References :


1) Avrameas  S., Coupling  of enzymes to protein with glutaraldehyde. Use of conjugates for the detection of antigen and antibodies, Immunochem.,6(1969)  43.


2) Catty D., Antibodies Volume II, A practical approach 106-108.