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ELISA Genie Technical Manual

Human PD-L1 / CD274 ELISA Kit

Catalogue Code: HUFI02708

Research Use Only

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1. Description and Principle

2. Key Features and Sample Types

3. Workflow Overview

4. Kit Contents

5. Shipping and Storage

6. Sample preparation

7. Protocol

8. Data Analysis

9. Important General Notes

1. Description and Principle

The ELISA Genie PD-L1 / CD274 ELISA Kit can assay for PD-L1 / CD274 in the following samples: serum,

blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our PD-L1 / CD274 ELISA Kits Work?

The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative

measurement of analytes in a wide variety of samples. As today’s scientists demand high quality

consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and

reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich

ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well

microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast

protocol meaning you achieve your results rapidly. So, once you have prepared and plated your

samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary

antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate

length of time. After washing the plate according to the protocol and addition of the TMB (3,3’,5,5’-

Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an

enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates

the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader

at 450nm. The amount of bound PD-L1 / CD274 is proportional to the signal generated by the reaction

meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

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2. Key features and Sample Types

Aliases:

PDCD1LG1/CD274/PDCD1L1/PDCD1LG1/PD-L1/B7-H/B7-H1/B7H1/CD274 antigen/CD274

molecule/B7 homolog 1/PDL1/PDCD1 ligand 1/programmed cell death 1 ligand 1/Programmed death

ligand 1

Detection method:

Sandwich

Range:

0.156-10ng/ml

Sensitivity:

0.094ng/ml

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3. Workflow Overview

Prepare reagents, samples and standards and equilibrate to room temperature

Wash plate x2 and aspirate off all liquid

Add 100µL standard, sample or blank to wells

Incubate @ 37⁰C for 90 minutes

Remove the liquid. Add 100µL Biotin-detection antibody work solution

Incubate @ 37⁰C for 60 minutes

Wash plate x 3

Add 100µL SABC working solution

Incubate @ 37⁰C for 30 minutes

Wash plate x 5

Add 90µL TMB Substrate Solution

Incubate @ 37⁰C for 15-30 minutes

Add 50µl Stop solution to each well

Measure OD @ 450nm

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4. Kit Contents

Product Size Cat. Code

Human PD-L1 / CD274 ELISA Kit 96 assays HUFI02708

Each kit contains reagents for 96 assays in a 96 well plate including:

No. Component Qty No. Component Qty

1 Standard 2 vials 7 HRP-Streptavidin Conjugate (SABC)

120µl

2 Sample/Standard Dilution buffer

20ml 8 Biotin-detection antibody (Concentrated)

120µl

3 ELISA Strip plate coated with monoclonal antibodies

12w×8s 9 Stop Solution 10ml

4 Wash Buffer (25×) 30ml 10 TMB Substrate 10ml

5 SABC dilution buffer 10ml 11 Plate Sealer 5 pieces

6 Antibody dilution buffer 10ml 12 Manual 1

Additional Materials required

1. 37⁰C incubator 2. Plate shaker 3. Plate Reader with 450nm filter 4. Precision pipettes and disposable pipette tips 5. Distilled water 6. Disposable tubes for sample dilution 7. Absorbent paper

5. Shipping and Storage

Store kit @ 4⁰C for 6 months

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6. Sample Preparation

General considerations: extract samples as soon as possible after specimen collection. According to

best practices, experiments should be carried out as soon as possible after the extraction.

Alternatively, extract can be kept at -20⁰C but for optimal results, avoid repeated freeze-thaw cycles.

Samples that contain NaN3 cannot be detected as it interferes with HRP.

Serum: Use a serum separator tube (SST) and allow samples to clot for 30 minutes before

centrifugation for 15 minutes at approximately 1000 × g. Remove serum and assay immediately or

aliquot and store samples at -20⁰C or -80⁰C.

Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes

at 1000 × g at 2⁰C - 8⁰C within 30 minutes of collection. Store samples at -20⁰C or -80⁰C. Avoid repeated

freeze-thaw cycles. Note: over hemolyzed samples are not suitable for use with this kit.

Urine: collect in a sterile container, centrifuge for 20 mins @ 2000-3000 rpm. Remove supernatant

and if any precipitation is detected repeat centrifugation step. A similar protocol can be used for

cerebrospinal fluid.

Cell culture supernatant: collect supernatant and centrifuge @ 4⁰C for 20 mins @ 2000-3000 rpm.

Remove supernatant and rinse cells x2 times with PBS (pH 7.2-7.4) and perform a total cell count.

Optimal cell concentration is 1 million / ml. To release cellular components, dilute the cell pellet in

PBS and use 3-4 freeze-thaw cycles in liquid Nitrogen (commercial lyses buffers can be used according

to manufacturer’s instructions). Centrifuge @ 4⁰C for 20 mins @ 2000-3000 rpm to pellet debris and

remove clear supernatant to clean microcentrifuge tube for ELISA or storage.

Tissue samples: the preparation of tissue homogenates will vary depending upon tissue type. For this

assay, tissue was rinsed with 1X PBS to remove excess blood, homogenized in 20mL of 1X PBS and

stored overnight at ≤ -20⁰C After two freeze-thaw cycles were performed to break the cell

membranes, the homogenates were centrifuged for 5 minutes at 5000 x g. Remove the supernatant

and assay immediately or aliquot and store at ≤ -20⁰C.

Notes:

1. Samples to be used within 5 days may be stored at 2-8⁰C, otherwise samples must be stored at -

20⁰C (1 month) or -80⁰C (2 months) to avoid loss of bioactivity and contamination.

2. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA

results due to the impacts of certain chemicals.

3. Influenced by the factors including cell viability, cell number and also sampling time, samples

from cell culture supernatant may not be detected by the kit

4. Sample hemolysis will influence the result, so hemolytic specimen cannot be detected.

5. When performing the assay slowly bring samples to room temperature.

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7. Protocol

Manual Washing

Discard the solution in the plate without touching the side walls. Clap the plate on absorbent filter

papers or other absorbent material. Fill each well completely with 350ul wash buffer and soak for 1

to 2 minutes, then aspirate contents from the plate, and clap the plate on absorbent filter papers or

other absorbent material. Repeat this procedure two more times for a total of THREE washes.

Automated Washing

Aspirate all wells, then wash plate THREE times with 350ul wash buffer. After the final wash, invert

plate, and clap the plate on absorbent filter papers or other absorbent material. It is recommended

that the washer be set for a soaking time of 1 minute.

Sample Collection and Storage

Isolate the test samples soon after collecting, then, analyze immediately (within 2 hours). Or aliquot

and store at -20°C for long term. Avoid multiple freeze-thaw cycles.

Serum: Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000×g. Collect the supernatant and carry out the assay immediately. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin.

Plasma: Collect plasma using EDTA-Na2 as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8°C within 30 minutes of collection. Collect the supernatant and carry out the assay immediately. Avoid hemolysis, high cholesterol samples.

Tissue homogenates: For general information, hemolysis blood may affect the result, so you should rinse the tissues with ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then minced to small pieces which will be homogenized in PBS (the volume depends on the weight of the tissue. 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitor is recommended to add into the PBS.) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifugated for 5minutes at 5000×g to get the supernatant.

Cell culture supernatant: Centrifuge supernatant for 20 minutes to remove insoluble impurity and cell debris at 1000×g at 2 - 8°C. Collect the clear supernatant and carry out the assay immediately.

Other biological fluids: Centrifuge samples for 20 minutes at 1000×g at 2 - 8°C. Collect the supernatant and carry out the assay immediately.

Sample preparation: Samples should be clear and transparent and be centrifuged to remove suspended solids.

Note: Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -

20°C (≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and contamination. Hemolyzed

samples are not suitable for use in this assay.

Sample Dilution Guideline

End user should estimate the concentration of the target protein in the test sample first, and select a

proper dilution factor to make the diluted target protein concentration falls the optimal detection

range of the kit. Dilute the sample with the provided dilution buffer, and several trials may be

necessary in practice. The test sample must be well mixed with the dilution buffer. And also standard

curves and sample should be make in pre-experiment.

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High target protein concentration (100-1000ng/ml): Dilution: 1:100. (i.e. Add 1µl of sample into 99 µl of Sample / Standard dilution buffer.)

Medium target protein concentration (10-100ng/ml): Dilution: 1:10.( i.e. Add 10 µl of sample into 90 µl of Sample / Standard dilution buffer.)

Low target protein concentration (0.156-10ng/ml): Dilution: 1:2.( i.e. Add 50 µl of sample into 50 µl of Sample / Standard dilution buffer.)

Very low target protein concentration (≤0.156ng/ml): Unnecessary to dilute, or dilute at 1:2.

Reagent Preparation and Storage

Bring all reagents to room temperature before use.

1. Wash Buffer:

Dilute 30mL of Concentrated Wash Buffer into 750 mL of Wash Buffer with deionized or distilled

water. Put unused solution back at 4°C. If crystals have formed in the concentrate, you can warm it

with 40°C water bath (Heating temperature should not exceed 50°C) and mix it gently until the

crystals have completely dissolved. The solution should be cooled to room temperature before use.

2. Standard:

1). 10ng/ml of standard solution: Add 1 ml of Sample / Standard dilution buffer into one Standard

tube, keep the tube at room temperature for 10 min and mix thoroughly.

2). 5ng/m --> 0.156ng/ml of standard solutions: Label 6 Eppendorf tubes with 5ng/ml, 2.5ng/ml,

1.25ng/ml, 0.625ng/ml, 0.3125ng/ml, 0.156ng/ml, respectively. Aliquot 0.3 ml of the Sample /

Standard dilution buffer into each tube. Add 0.3 ml of the above 10ng/ml standard solution into 1st

tube and mix thoroughly. Transfer 0.3 ml from 1st tube to 2nd tube and mix thoroughly. Transfer 0.3

ml from 2nd tube to 3rd tube and mix thoroughly, and so on.

0.3 ml 0.3 ml 0.3 ml 0.3 ml 0.3 ml 0.3 ml

Blank

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2.5

1.25

0.625

ng/ml

0.3125

0.156

Note: The standard solutions are best used within 2 hours. The standard solution should be at 4°C

for up to 12 hours. Or store at -20 °C for up to 48 hours. Avoid repeated freeze-thaw cycles.

3. Preparation of Biotin- detection Antibody working solution

Prepare within 1 hour before the experiment.

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1) Calculate the total volume of the working solution: 0.1 ml / well × quantity of wells. (Allow 0.1-0.2 ml more than the total volume)

2) Dilute the Biotin- detection antibody with Antibody dilution buffer at 1:100 and mix thoroughly. (i.e. Add 1 µl of Biotin- detection antibody into 99 µl of Antibody dilution buffer.)

4. Preparation of HRP-Streptavidin Conjugate (SABC) working solution:

Prepare within 30min before the experiment.

1) Calculate the total volume of the working solution: 0.1 ml / well × quantity of wells. (Allow 0.1-0.2 ml more than the total volume)

2) Dilute the SABC with SABC dilution buffer at 1:100 and mix thoroughly. (i.e. Add 1 µl of SABC into 99 µl of SABC dilution buffer.)

Assay Procedure

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min

at room temperature (37 °C). When diluting samples and reagents, they must be mixed completely

and evenly. It is recommended to plot a standard curve for each test.

1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then,

record their positions. It is recommended to measure each standard and sample in duplicate. Wash

plate 2 times before adding standard, sample and control (zero) wells!

2. Aliquot 0.1ml of 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml, 0.3125ng/ml, 0.156ng/ml,

standard solutions into the standard wells.

3. Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.

4. Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other

biological fluids.) into test sample wells.

5. Seal the plate with a cover and incubate at 37 °C for 90 min.

6. Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or

other absorbent material. Do NOT let the wells completely dry at any time. Do Not Wash Plate!

7. Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test

sample & zero wells). Add the solution at the bottom of each well without touching the side wall.

8. Seal the plate with a cover and incubate at 37°C for 60 min.

9. Remove the cover, and wash plate 3 times with Wash buffer.

10. Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30

min.

11. Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer

stay in the wells for 1-2 min.

12. Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within

15-30 min. (Note: This incubation time is for reference use only, the optimal time should be

determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most

concentrated PDCD1LG1 standard solutions), the other wells show no obvious color.

13. Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow

immediately.

14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop

solution.

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For calculation, (the relative O.D.450) = (the O.D.450 of each well) – (the O.D.450 of Zero well). The

standard curve can be plotted as the relative O.D.450 of each standard solution (Y) vs. the respective

concentration of the standard solution (X). The PDCD1LG1 concentration of the samples can be

interpolated from the standard curve. Recommended to use professional software curve expert to

1.3.

Note: If the samples measured were diluted, multiply the dilution factor to the concentrations from

interpolation to obtain the concentration before dilution.

Summary

1. Wash plate 2 times before adding standard, sample and control (zero) wells.

2. Add 100µL standard or sample to each well for 90 minutes at 37°C

3. Add 100µL Biotin- detection antibody working solution to each well for 60 minutes at 37°C

4. Aspirate and wash 3 times

5. Add 100µL SABC working solution to each well. Incubate for 30 minutes at 37°C

6. Aspirate and wash 5 times

7. Add 90µL TMB substrate. Incubate 15 -30 minutes at 37°C

8. Add 50µL Stop Solution. Read at 450nm immediately

9. Calculation of results

Typical Data & Standard Curve

Results of a typical standard run of a PDCD1LG1 ELISA Kit are shown below. This standard curve was

generated at our lab for demonstration purpose only. Each user should obtain their own standard

curve as per experiment. (N/A=not applicable)

X ng/ml 0 0.156 0.312 0.625 1.25 2.5 5 10

Y OD450 0.012 0.094 0.178 0.399 0.551 0.913 1.616 2.319

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Specificity

This assay has high sensitivity and excellent specificity for detection of PDCD1LG1 . No significant

cross-reactivity or interference between PDCD1LG1 and analogues was observed.

Note: Limited by current skills and knowledge, it is impossible for us to complete the cross- reactivity

detection between PDCD1LG1 and all the analogues, therefore, cross reaction may still exist.

Recovery

Matrices listed below were spiked with certain level of PDCD1LG1 and the recovery rates were

calculated by comparing the measured value to the expected amount of PDCD1LG1 in samples.

Matrix Recovery range (%) Average(%)

serum(n=5) 86-102 93

EDTA plasma(n=5) 86-103 94

heparin plasma(n=5) 91-105 98

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of

PDCD1LG1 and their serial dilutions. The results were demonstrated by the percentage of calculated

concentration to the expected.

Sample 1:2 1:4 1:8 1:16

serum(n=5) 87-101% 87-102% 87-105% 85-102%

EDTA plasma(n=5) 82-100% 93-101% 90-101% 83-101%

heparin plasma(n=5) 81-96% 80-92% 82-96% 80-100%

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Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level

PDCD1LG1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level

PDCD1LG1 were tested on 3 different plates, 8 replicates in each plate.

CV (%) = SD/meanX100

Intra-Assay: CV<8%

Inter-Assay: CV<10%

Stability

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than

10% within the expiration date under appropriate storage condition.

Standard(n=5) 37°C for 1 months 4°C for 6 months

Average(%) 80 95-100

To minimize extra influence on the performance, operation procedures and lab conditions,

especially room temperature, air humidity, incubator temperature should be strictly controlled. It is

also strongly suggested that the whole assay is performed by the same operator from the beginning

to the end.

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9. Important General Notes 1. The final experimental results will be closely related to validity of the products, laboratory skills of

the end-users and the experimental conditions. Please make sure that sufficient samples are available.

2. Kits from different batches may be a little different in detection range, sensitivity and color developing time. Please perform the experiment exactly according to the instructions.

3. There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results.

4. Do not remove microtiter plate from the storage bag until needed.

5. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

6. Use fresh disposable pipette tips for each liquid transfer to avoid contamination.

7. Do not substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.

8. In order to achieve reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before every assay for each batch is recommended.

9. Each kit has been strictly passed Q.C tested. However, results from end-users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipment. Intra-assay variance among kits from different batches might arise from aforementioned factors.

Safety Precaution

1. The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

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Contact Details

Dublin, Ireland

Email: info@elisagenie.com

Web: www.elisagenie.com

Copyright © 2017 ReagentBio, All Rights Reserved. All information / detail is correct at time of going to print.