Y GeneTel Laboratories LLC

Leader in Proteomics Abs and Abs Arrays1202 Ann Street, Madison, WI 53713 Phone 877-248-4316 Fax 608-257-1385 Email info@genetel-lab.com

A1 ELISA Kit Protocol
Received day: ____________________Assay Date:_______________________
Scientist:____________________
Description: To measure A1 beta casein level in milk samples using a direct ELISA
kit. A1 beta casein standards (2.5ng/ml-160ng/ml) and diluted milksamples will be coated on the high binding plate and the A1 specificchicken pAbs will be used to detect the A1 beta casein. Rabbit antichicken IgY HRP conjugate will be used for the signal amplification.
After color development, OD450 will be measured and A1 standard curve
will be established. Concentration of A1 beta casein in tested sample willbe estimated based on the standard curve. In general, house milk sample
need to be diluted to 1:200,000 to 1:500,000. For each plate, two columns
will be used for standard curve establishment with the detecting range of
2.5ng/ml to 160ng/ml A1.
Materials provided in the kit:
A1 beta casein standard, 40ug/ml (40ng/ul), -20oC
Chicken Anti-A1 beta casein specific pAbs, 0.5mg/ml, -20oC
Rabbit Anti-Chicken IgY HRP conjugate, use at 1:1000 dilution, -20oC
Binding Buffer for standard and milk samples: 100ml
10X TBS 100ml, add 900ml D.I. water to make 1000ml TBS
Blocking Buffer: 1.1 gram BSA, add 20.5ml TBS
Ab dilution buffer: 1% BSA in TBST: 1g BSA, add 100ml TBST
Tween-20: 1ml 50%, add into 900ml TBS to make TBST
Substrate TMB, 6ml
ELISA Plate, 96-well
ELISA Record Sheet
Materials customer needs and prepare:
ELISA Plate Washer and Plate Shaker
ELISA plate Reader
Pipettors and tips
HCl, 1M for stopping reaction in final step
NaOH, 0.1M for milk sample preparation
Buffer Preparation:
TBS: add the 100ml 10X TBS into 900ml D.I. water and mix well
Blocking buffer: Tube contains 1.1 gram BSA in TBS, add 20.5ml TBS
TBST: add one tube of provided 50% Tween-20 into the 900ml TBS
Ab dilution buffer: 1% BSA in TBST: 1g BSA in 100ml TBST.

GeneTel Laboratories LLC

Leader in Proteomics Abs and Abs Arrays1202 Ann Street, Madison, WI 53713 Phone 877-248-4316 Fax 608-257-1385 Email info@genetel-lab.com

Procedures:
1.

Prepare A1 standard in seven 1.5ml tubes. In the first tube (tube#1), add 4ul of40ug/ml A1 into 996ul Binding Buffer to make 160ng/ml A1 standard. Add
500ul Binding Buffer into each of the rest 6 tubes and do a serial 2X dilution
by taking out 500ul from the first tube (with 160ng/ml A1), so that tube#1 has160ng/ml, tube#2: 80ng/ml, #3: 40ng/ml; #4: 20ng/ml; #5: 10ng/ml; #6:5ng/ml; #7: 2.5ng/ml. Immediately transfer 100ul/well into ELISA plateafter dilution.

2.

Prepare the tested milk samples by diluting milk in Binding Buffer
accordingly. In general, caseins are not soluble in neutral pH and thus milk
sample needs to be diluted at 1:200 to 1:500 in 0.5M NaOH first, then, further
dilute in Binding Buffer to obtain 1:200,000-1:500,000. Immediately apply
the diluted samples in Binding Buffer to ELISA plate at 100ul/well. Keep theplate at room temperature for one hour and store the plate at 4 oC overnight to3 days.

3.

Empty coating solution from the wells and block with 200ul/well Blocking
buffer for one hour at room temperature. All the following steps will be
performed at room temperature.

4.

Empty blocking solution from the wells and wash 3X with TBST.

5.

While washing the plate, prepare (10ml per plate) A1 specific detecting pAb(OA0101) in Ab dilution buffer at 1:2000 dilution, ie, add 5ul A1 Detecting
Abs into 10ml Ab dilution buffer.

6.

Add the above prepared A1 detecting pAb at 100ul/well to the plate and shakefor 2 hour at RT.

7.

Wash 3X with TBST.

8.

While washing the plate, prepare Rabbit Anti-Chicken IgY HRP (RCYHRP)
conjugate (10ml per plate) solution: dilute the HRP conjugate to 1:1000 in Ab
dilution buffer (10ul HRP conjugate into 10ml Ab dilution buffer, mix well).
Add 100ul/well to the plate.

9.

Mix the plate on the shaker for another 1 hour at RT.

10.

Wash 5X with TBST.

11.

Add 50ul/well substrate (TMBS). Let the color to develop 3-5 minutes.

12.

Stop the reaction with 50ul 1M HCl. and read OD450 immediately.

GeneTel Laboratories LLC

Leader in Proteomics Abs and Abs Arrays1202 Ann Street, Madison, WI 53713 Phone 877-248-4316 Fax 608-257-1385 Email info@genetel-lab.com

13.

Record the development time on the ELISA recording sheet along withOD450 readout.

14.

Make a standard curve using the average OD450 from the two reference
standards for each point. Determine the A1 concentration for the tested
samples based on their OD450 and standard curve. make sure that the final
concentration is obtained by multiplying with dilution factor. Example:
standard curve with 3 minutes color development.