RayBio Mouse IFN-gamma ELISA Kit User Manual

®

RayBio Mouse IFN-gamma ELISA

Kit

Catalog #: ELM-IFNg

User Manual

Last revised January 17, 2022

Caution:

Extraordinarily useful information enclosed

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RayBiotech, Inc.

________________________________________

RayBio

®

Mouse IFN-gamma ELISA Kit Protocol

Table of Contents

Section

I.

II.

III.

IV.

V.

VI.

VII.

VIII.

Introduction

Storage

Reagents

Additional Materials Required

Reagent Preparation

Assay Procedure

Assay Procedure Summary

Calculation of Results

A. Typical Data

B. Sensitivity

C. Spiking & Recovery

D. Linearity

E. Reproducibility

Specificity

Troubleshooting Guide

Page #

3

4

4

4

5

6

7

8

8

8

9

9

10

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IX.

X.

Please read the entire manual carefully before starting your experiment

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I. INTRODUCTION

IFN-gamma is produced mainly by T-cells and natural killer cells activated by antigens,

mitogens, or alloantigens. It is produced by lymphocytes expressing the surface antigens

CD4 and CD8. Mouse IFN-gamma is a polypeptide of 136 amino acids, containing four exons

and three introns. It plays an important role in the immune IFN-gamma response. IFN-gamma

is a modulator of T-cell growth and functional differentiation. It is a growth-promoting factor for

T-lymphocytes and potentiates the response of these cells to mitogens or growth factors.

The RayBio

®

Mouse IFN-gamma ELISA kit is an in vitro enzyme-linked immunosorbent

assay for the quantitative measurement of mouse IFN-gamma in serum, plasma and cell

culture supernatants. This assay employs an antibody specific for mouse IFN-gamma coated

on a 96-well plate. Standards and samples are pipetted into the wells and IFN-gamma

present in a sample is bound to the wells by the immobilized antibody. The wells are washed

and biotinylated anti-mouse IFN-gamma antibody is added. After washing away unbound

biotinylated antibody, HRP- conjugated streptavidin is pipetted to the wells. The wells are

again washed, a TMB substrate solution is added to the wells and color develops in

proportion to the amount of IFN-gamma bound. The Stop Solution changes the color from

blue to yellow, and the intensity of the color is measured at 450 nm.

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II. STORAGE

The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid

repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended

storage, it is recommended to store at -80°C. For prepared reagent storage, see table below.

III. REAGENTS

Component

IFN-gamma Microplate (Item

A)

Wash Buffer Concentrate

(20X) (Item B)

Standard Protein (Item C)

Detection Antibody IFN-

gamma (Item F)

HRP-Streptavidin

Concentrate (Item G)

TMB One-Step Substrate

Reagent (Item H)

Stop Solution (Item I)

Assay Diluent A (Item D)

Assay Diluent B (Item E)

Size / Description

96 wells (12 strips x 8 wells) coated with anti-

Mouse IFN-gamma.

25 ml of 20X concentrated solution.

2 vials of Mouse IFN-gamma. 1 vial is enough to

run each standard in duplicate.

2 vials of biotinylated anti-Mouse IFN-gamma.

Each vial is enough to assay half the microplate.

200 µl 440X concentrated HRP-conjugated

streptavidin.

12 ml of 3,3,5,5'-tetramethylbenzidine (TMB) in

buffer solution.

8 ml of 0.2 M sulfuric acid.

30 ml of diluent buffer, 0.09% sodium azide as

preservative.

15 ml of 5X concentrated buffer.

Storage / Stability

After Preparation

1 month at 4°C*

1 month at 4°C

1 week at -80°C

5 days at 4°C

Do not store and

reuse.

N/A

N/A

N/A

1 month at 4°C

*Return unused wells to the pouch containing desiccant pack, reseal along entire edge.

IV. ADDITIONAL MATERIALS REQUIRED

1.

2.

3.

4.

5.

6.

7.

8.

Microplate reader capable of measuring absorbance at 450 nm.

Precision pipettes to deliver 2 µl to 1 ml volumes.

Adjustable 1-25 ml pipettes for reagent preparation.

100 ml and 1 liter graduated cylinders.

Absorbent paper.

Distilled or deionized water.

Log-log graph paper or computer and software for ELISA data analysis.

Tubes to prepare standard or sample dilutions.

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V. REAGENT PREPARATION

1.

Bring all reagents and samples to room temperature (18 - 25ºC) before use.

2.

Assay Diluent B (Item E) should be diluted 5-fold with deionized or distilled water before

use.

3.

Sample dilution: Assay Diluent A (Item D) should be used for dilution of serum and

plasma samples. 1X Assay Diluent B (Item E) should be used for dilution of cell culture

supernatant samples. The suggested dilution for normal serum/plasma is 2 fold.

Note: Levels of IFN-gamma may vary between different samples. Optimal dilution

factors for each sample must be determined by the investigator.

4.

Preparation of standard: Briefly spin a vial of Item C. Add 400 µl Assay Diluent A (for

serum/plasma samples) or 1X Assay Diluent B (for cell culture medium) into Item C vial

to prepare a 20 ng/ml standard. Dissolve the powder thoroughly by a gentle mix. Add 50

µl IFN-gamma standard from the vial of Item C, into a tube with 450 µl Assay Diluent A

or 1X Assay Diluent B to prepare a 2,000 pg/ml stock standard solution. Pipette 400 µl

Assay Diluent A or 1X Assay Diluent B into each tube. Use the stock standard solution

to produce a dilution series (shown below). Mix each tube thoroughly before the next

transfer. Assay Diluent A or 1X Assay Diluent B serves as the zero standard (0 pg/ml).

50 µl200 µl200 µl200 µl200 µl200 µl200 µl

Item C +

400 µl

Std1

450 µl

Std2

400 µl

Std3

400 µl

Std4

400 µl

Std5

400 µl

Std6

400 µl

Std7

400 µl

Zero

Standard

400 µl

Diluent

volume

Conc.

20 ng/ml

2000

pg/ml

666.7

pg/ml

222.2

pg/ml

74.07

pg/ml

24.69

pg/ml

8.23

pg/ml

2.74

pg/ml

0 pg/ml

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5.

If the Wash Concentrate (20X) (Item B) contains visible crystals, warm to room

temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into

deionized or distilled water to yield 400 ml of 1X Wash Buffer.

6.

Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µl of 1X Assay

Diluent B (Item E) into the vial to prepare a detection antibody concentrate. Pipette up

and down to mix gently (the concentrate can be stored at 4ºC for 5 days). The detection

antibody concentrate should be diluted 120-fold with 1X Assay Diluent B (Item E) and

used in step 5 of Part VI Assay Procedure.

7.

Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to

mix gently before use, as precipitates may form during storage. HRP-Streptavidin

concentrate should be diluted 440-fold with 1X Assay Diluent B (Item E).

For example: Briefly spin the vial (Item G) and pipette up and down to mix gently. Add

25 µl of HRP-Streptavidin concentrate into a tube with 11 ml 1X Assay Diluent B to

prepare a 440-fold diluted HRP-Streptavidin solution (don't store the diluted solution for

next day use. Mix well.

VI. ASSAY PROCEDURE

1.

Bring all reagents and samples to room temperature (18 - 25ºC) before use. It is

recommended that all standards and samples be run at least in duplicate.

2.

Label removable 8-well strips as appropriate for your experiment.

3.

Add 100 µl of each standard (see Reagent Preparation step 3) and sample into

appropriate wells. Cover wells and incubate for 2.5 hours at room temperature with

gentle shaking.

4.

Discard the solution and wash 4 times with 1X Wash Solution. Wash by filling each well

with Wash Buffer (300 µl) using a multi-channel Pipette or autowasher. Complete

removal of liquid at each step is essential to good performance. After the last wash,

remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot

it against clean paper towels.

5.

Add 100 µl of 1X prepared biotinylated antibody (Reagent Preparation step 6) to each

well. Incubate for 1 hour at room temperature with gentle shaking.

6.

Discard the solution. Repeat the wash as in step 4.

7.

Add 100 µl of prepared Streptavidin solution (see Reagent Preparation step 7) to each

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well. Incubate for 45 minutes at room temperature with gentle shaking.

8.

Discard the solution. Repeat the wash as in step 4.

9.

Add 100 µl of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30

minutes at room temperature in the dark with gentle shaking.

10.

Add 50 µl of Stop Solution (Item I) to each well. Read at 450 nm immediately.

VII. ASSAY PROCEDURE SUMMARY

1.

Prepare all reagents, samples and standards as instructed.

2.

Add 100 µl standard or sample to each well. Incubate 2.5 hours at room temperature.

3.

Add 100 µl prepared biotin antibody to each well. Incubate 1 hour at room temperature.

4.

Add 100 µl prepared Streptavidin solution. Incubate 45 minutes at room temperature.

5.

Add 100 µl TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at

room temperature.

6.

Add 50 µl Stop Solution to each well. Read at 450 nm immediately.

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VIII. CALCULATION OF RESULTS

Calculate the mean absorbance for each set of duplicate standards, controls and samples,

and subtract the average zero standard optical density. Plot the standard curve on log-log

graph paper or using Sigma plot software, with standard concentration on the x-axis and

absorbance on the y-axis. Draw the best-fit straight line through the standard points.

A. TYPICAL DATA

These standard curves are for demonstration only. A standard curve must be run with each

assay.

B. SENSITIVITY

The minimum detectable dose of Mouse IFN-gamma was determined to be 1.3 pg/ml.

Minimum detectable dose is defined as the analyte concentration resulting in an absorbance

that is 2 standard deviations higher than that of the blank (diluent buffer).

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C. SPIKING & RECOVERY

Recovery was determined by spiking various levels of Mouse IFN-gamma into the sample

types listed below. Mean recoveries are as follows:

D. LINEARITY

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E. REPRODUCIBILITY

Intra-Assay CV%: <10%

Inter-Assay CV%: <12%

IX. SPECIFICITY

This ELISA kit shows no cross-reactivity with any of the cytokines tested: Mouse CD30, L

CD30, T CD40, CRG-2, CTACK, CXCL16, Eotaxin , Eotaxin-2, Fas Ligand, Fractalkine,

GCSF, GM-CFS, IGFBP-3, IGFBP-5, IGFBP-6, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-3 Rb, IL-4,

IL-5, IL-6, IL-9, IL-10, IL-12 p40/p70, IL-12 p70, IL-13, IL-17, KC, Leptin R, Leptin (OB), LIX, L-

Selectin, Lymphotactin, MCP-1, MCP-5, M-CSF, MIG, MIP-1 alpha, MIP-1 gamma, MIP-2,

MIP-3 beta, MIP-3 alpha, PF-4, P-Selectin, RANTES, SCF, SDF-1 alpha, TARC, TCA-3,

TECK, TIMP-1, TNF-alpha, TNF RI, TNF RII, TPO, VCAM-1, VEGF.

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X. TROUBLESHOOTING GUIDE

Problem

Poor standard

curve

Cause

Inaccurate pipetting

Improper standard

dilution

Solution

Check pipettes

Briefly centrifuge Item C and dissolve

the powder thoroughly by gently mixing

Low signal

Improper preparation of

standard and/or

biotinylated antibody

Too brief incubation

times

Inadequate reagent

volumes or improper

dilution

Briefly spin down vials before opening.

Dissolve the powder thoroughly.

Ensure sufficient incubation time. Assay

procedure step 3 may be done

overnight at 4°C with gentle shaking

(note: may increase overall signals

including background).

Check pipettes and ensure correct

preparation

Large CV

Inaccurate pipetting

Air bubbles in wells

Check pipettes

Remove bubbles in wells

High

background

Plate is insufficiently

washed

Contaminated wash

buffer

Review the manual for proper wash. If

using a plate washer, ensure that all

ports are unobstructed.

Make fresh wash buffer

Low sensitivity

Improper storage of the

ELISA kit

Stop solution

Store your standard at <-70ºC after

reconstitution, others at 4ºC. Keep

substrate solution protected from light.

Add stop solution to each well before

reading plate

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®

ELISA Kits

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