Mouse Total Immunoglobulin (TIG)

Elisa kit

96 Tests
Catalog Number: MBS726928
Store all reagents at 2-8°C
Valid Period: six months

For samples:
Serum, plasma, cell culture supernatants, body fluid
and tissue homogenate

FOR RESEARCH USE ONLY!

NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!

PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!

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FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.
Table of Contents
12) Incomplete washing will adversely affect the test outcome. All
1. INTENDED USE ............................................................................... 3 washing must be performed with Wash Solution provided. All
residual wash liquid must be drained from the wells by efficient
2. PRINCIPLE OF THE ASSAY ......................................................... 3
aspiration or by decantation followed by tapping the plate forcefully
3. MATERIALS ..................................................................................... 4 on absorbent paper. Never insert absorbent paper directly into the
wells.
4. SPECIMEN COLLECTION AND STORAGE .............................. 5 13) Because TMB is light sensitive, avoid prolonged exposure to light.
Also avoid contact between TMB and metal, otherwise color may
5. MATERIALS AND EQUIPMENT REQUIRED BUT NOT
develop.
SUPPLIED ......................................................................................... 7

6. SAMPLE PREPARATION .............................................................. 7

7. REAGENTS PREPARATION ......................................................... 8

8. ASSAY PROCEDURE ...................................................................... 9

9. CALCULATION OF RESULTS .................................................... 10

10. CERTIFICATE OF ANALYSIS .................................................... 12

11. SAFETY NOTES ............................................................................. 13

12. QUALITY CONTROL ................................................................... 14

13. CONTACT US ................................................................................. 16

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FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.
12. QUALITY CONTROL 1. INTENDED USE

1) It is recommended that all standards, controls and samples be run in This TIG ELISA kit is a 1.5 hour solid-phase ELISA designed for the
duplicate. Standards and samples must be assayed at the same time. quantitative determination of Mouse TIG. This ELISA kit for research use
2) The coefficient of determination of the standard curve should be ≥ only, not for therapeutic or diagnostic applications!
0.95 and the highest O.D. should be more than 1.0.
3) Cover or cap all kit components and store at 2-8° C when not in use. 2. PRINCIPLE OF THE ASSAY
4) Microtiter plates should be allowed to come to room temperature
TIG ELISA kit applies the competitive enzyme immunoassay technique
before opening the foil bags. Once the desired number of strips has
utilizing a monoclonal anti-TIG antibody and an TIG-HRP conjugate. The
been removed, immediately reseal the bag with desiccants and store
assay sample and buffer are incubated together with TIG-HRP conjugate in
at 2-8°C to maintain plate integrity.
pre-coated plate for one hour. After the incubation period, the wells are
5) Samples should be collected in pyrogen/endotoxin-free tubes.
decanted and washed five times. The wells are then incubated with a
6) Samples should be frozen if not analyzed shortly after collection.
substrate for HRP enzyme. The product of the enzyme-substrate reaction
Avoid multiple freeze-thaw cycles of frozen samples. Thaw
forms a blue colored complex. Finally, a stop solution is added to stop the
completely and mix well prior to analysis.
reaction, which will then turn the solution yellow. The intensity of color is
7) When possible, avoid use of badly hemolyzed or lipemic serum. If
measured spectrophotometrically at 450nm in a microplate reader. The
large amounts of particulate matter are present, centrifuge or filter
intensity of the color is inversely proportional to the TIG concentration
prior to analysis.
since TIG from samples and TIG-HRP conjugate compete for the anti-TIG
8) When pipetting reagents, maintain a consistent order of addition from
antibody binding site. Since the number of sites is limited, as more sites are
well-to-well. This ensures equal incubation times for all wells.
occupied by TIG from the sample, fewer sites are left to bind TIG-HRP
9) Do not mix or interchange different reagent lots from various kit lots.
conjugate. A standard curve is plotted relating the intensity of the color
10) Do not use reagents after the kit expiration date.
(O.D.) to the concentration of standards. The TIG concentration in each
11) Read absorbance immediately after adding the stop solution.
sample is interpolated from this standard curve.

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FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.
3. MATERIALS cross-reactivity detection between TIG and all the analogues, therefore,
cross reaction may still exist in some cases.
All reagents provided are stored at 2-8° C. Refer to the expiration date on
the label.
11. SAFETY NOTES

MATERIALS SPECIFICATION QUANTITY 1) This kit contains small amount of 3, 3’, 5, 5’-Tetramethylbenzidine
1 MICROTITER PLATE 96 wells stripwell (TMB) in Substrate B. TMB is non-carcinogenic but it is hhazardous
2 ENZYME CONJUGATE 6.0 mL 1 vial in case of skin contact, eye contact, ingestion and inhalation. In case
3 STANDARD A 0 mg/ml 1 vial of contact, rinse affected area with plenty of water.
4 STANDARD B 1.0 mg/ml 1 vial 2) The Stop Solution provided with this kit is an acid solution. Wear
5 STANDARD C 2.5 mg/ml 1 vial protective gloves, clothing, and face protection.
6 STANDARD D 5.0 mg/ml 1 vial 3) Care should be taken when handling the Standard because of the
7 STANDARD E 10 mg/ml 1 vial known and unknown effects of it.
8 STANDARD F 25 mg/ml 1 vial 4) Care should also be taken to avoid contact of skin or eyes with other
9 SUBSTRATE A 6 mL 1 vial kit reagents or specimens. In the case of contact, wash immediately
10 SUBSTRATE B 6 mL 1 vial with water.
11 STOP SOLUTION 6 mL 1 vial 5) Do not pipette by mouth.
12 WASH SOLUTION (100 x) 10 mL 1 vial 6) Avoid generation of aerosols.
13 BALANCE SOLUTION 3 mL 1 vial 7) Waste must be disposed of in accordance with federal, state and local
14 INSTRUCTION 1 environmental control regulations.
8) All blood components and biological materials should be handled as
NOTE: The BALANCE SOLUTION is used only when the sample is cell potentially hazardous. Decontaminate and dispose specimens and all
culture supernatants, body fluid and tissue homogenate; if the sample is potentially contaminated materials as they could contain infectious
serum or plasma, then the BALANCE SOLUTION is a superfluous agents. The preferred method of decontamination is autoclaving for a
reagent. minimum of 1 hour a 121.5°C.

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FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.
NOTE:
1) Any variation in operator, pipetting and washing technique,
incubation time or temperature, and kit age can cause variation in The types of sample:
result. Each user should obtain their own standard curve. Sample I: serum or plasma
2) If samples have been diluted, the concentration read from the Sample II: cell culture supernatants, body fluid and tissue homogenate
standard curve must be multiplied by the dilution factor.
3) If specimens generate values higher than the highest standard, dilute
4. SPECIMEN COLLECTION AND STORAGE
the specimens and repeat the assay.
Serum - Use a serum separator tube and allow samples to clot for 2 hours
10. CERTIFICATE OF ANALYSIS at room temperature or overnight at 2-8°C. Centrifuge at approximately
1000 × g (or 3000 rpm) for 15 minutes. Remove serum and assay
1) In the same lot CV%:<10 immediately or aliquot and store samples at -20°C or -80°C.
2) Different lot CV%: <10
3) Spike Recovery: 92-101% Plasma - Collect plasma using EDTA or heparin as an anticoagulant.
4) Linearity: Centrifuge samples for 15 minutes at 1000 × g (or 3000 rpm) at 2 - 8°C
within 30 minutes of collection. Assay immediately or aliquot and store
Range %
samples at -20°C or -80°C.
1:2 94 – 102
1:4 92 - 104
Tissue homogenates - The preparation of tissue homogenates will vary
1:8 98 - 105
depending upon tissue type. For this assay, tissues were rinsed in ice-cold
1:16 94 - 109
PBS (0.02mol/L, pH 7.0-7.2) to remove excess blood thoroughly and
5) Sensitivity: The sensitivity in this assay is 0.1 mg/ml. weighed before homogenization. Minced the tissues to small pieces and
6) Specificity: This assay has high sensitivity and excellent specificity homogenized them in a certain amount of PBS with a glass homogenizer
for detection of TIG. No significant cross-reactivity or interference on ice. The resulting suspension was subjected to ultrasonication or to two
between TIG and analogues was observed. NOTE: Limited by freeze-thaw cycles to further break the cell membranes. After that, the
current skills and knowledge, it is impossible for us to complete the homogenates were centrifugated for 15 minutes at 1500×g (or 5000 rpm).

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FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.
Remove the supernate and assay immediately or aliquot and store samples 1) The standard curve is used to determine the amount of samples.
at -20°C or -80°C. 2) First, average the duplicate readings for each standard and sample.
All O.D. values are subtracted by the mean value of blank control
Cell lysates - Cells should be lysed according to the following directions. before result interpretation. DO NOT subtract the O.D of standard
1) Adherent cells should be detached with trypsin and then collected by zero.
centrifugation. Suspension cells can be collected by centrifugation 3) Construct a standard curve by plotting the average O.D. for each
directly. standard on the vertical (Y) axis against the concentration on the
2) Wash cells three times in PBS. horizontal (X) axis, and draw a best fit curve e using graph paper or
3) Cells were resuspended in PBS and subjected to ultrasonication for 3 statistical software to generate a four paramater logistic (4-PL)
times. Alternatively, freeze cells at -20 °C. Thaw cells with gentle curve-fit or logit-log linear regression curve. An x-axis for the optical
mixing. Repeat the freeze/thaw cycle for 3 times. density and a y-axis for the concentration is also a choice. The data
4) Centrifuge at 1000×g (or 3000 rpm) for 15 minutes at 2-8 °C to may be linearized by plotting the log of the concentrations versus the
remove cellular debris. log of the O.D. and the best fit line can be determined by regression
5) Assay immediately or store samples at -20°C or -80°C. analysis.
4) Calculate the concentration of samples corresponding to the mean
Cell culture supernatants and other body fluids - Centrifuge cell culture absorbance from the standard curve.
media at 1000 × g (or 3000 rpm) for 15 minutes to remove debris. Assay 5) Standard curve for demonstration only.
immediately or store samples at -20°C or -80°C. S = 0.05195277
r = 0.99598579
3
1.5
NOTE:
7
1.2
1) Samples should be aliquoted and must be stored at -20°C (less than 3

Y Axis (units)
2
months) or -80°C (less than 6 months) to avoid loss of bioactivity and 1.0

contamination. If samples are to be run within 24 hours, they may be 0.7

7

stored at 2- 8°C. Avoid repeated freeze-thaw cycles. 0.5

2

2) Prior to assay, the frozen sample should be brought to room 6

0.2
temperature slowly and mixed gently.
1
0.0
3.9 15.8 27.7 39.6 51.5 63.5 75.4

X Axis (units)
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FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.
4) Wash the microtiter plate using one of the specified methods 3) Tissue or cell extraction samples prepared by chemical lysis buffer
indicated below: may cause unexpected ELISA results due to the impacts of certain
a) Manual Washing: Remove incubation mixture by aspirating contents chemicals.
of the plate into a sink or proper waste container. Fill in each well 4) Samples containing a visible precipitate must be clarified prior to use
completely with 1× wash solution, and then aspirate contents of the in the assay. Care should be taken to minimize hemolysis. Do not use
plate into a sink or proper waste container. Repeat this procedure five grossly hemolyzed or lipemic specimens.
times for a total of FIVE washes. After washing, invert plate, and 5) Do not use heat-treated specimens.
blot dry by hitting the plate onto absorbent paper or paper towels
until no moisture appears. Note: Hold the sides of the plate frame 5. MATERIALS AND EQUIPMENT REQUIRED BUT NOT
firmly when washing the plate to assure that all strips remain SUPPLIED
securely in frame. Complete removal of liquid at each step is
essential to good performance. 1) Precision pipettors and disposable tips to deliver 10-1000 µl. A
b) Automated Washing: Wash plate FIVE times with diluted wash multi-channel pipette is desirable for large assays.
solution (350-400 µL/well/wash) using an auto washer. After 2) 100 mL and 1 liter graduated cylinders.
washing, dry the plate as above. It is recommended that the washer 3) Distilled or deionized water.
be set for a soaking time of 10 seconds and shaking time of 5 seconds 4) Tubes to prepare sample dilutions.
between each wash. 5) Absorbent paper.
5) Add 50 µL Substrate A and 50 µL Substrate B to each well 6) Microplate reader capable of measuring absorbance at 450 nm.
including blank control well, subsequently. Cover and incubate for 7) Centrifuge capable of 3000 × g.
10-15 minutes at 20-25°C. (Avoid sunlight). 8) Microplate washer or washing bottle.
6) Add 50 µL of Stop Solution to each well including blank control 9) Incubator (37°C).
well. Mix well. 10) Data analysis and graphing software.
7) Determine the Optical Density (O.D.) at 450 nm using a microplate
reader immediately. 6. SAMPLE PREPARATION

9. CALCULATION OF RESULTS 1) We are only responsible for the kit itself, but not for the samples
consumed during the assay. The user should calculate the

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FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.
 possible amount of the samples used in the whole test. Please reserve dilution. PBS (pH 7.0-7.2) or 0.9% physiological saline can be used
sufficient amount of samples in advance. as dilution buffer.
2) Please predict the concentration before assaying. If values for these 16) Wash Solution - Dilute 10 mL of Wash Solution concentrate (100×)
are not within the range of the standard curve, users must determine with 990 mL of deionized or distilled water to prepare 1000 mL of
the optimal sample dilutions for their particular experiments. Wash Solution (1×). If crystals have formed in the concentrate, warm
3) If the samples are not indicated in the manual, a preliminary to room temperature and mix gently until the crystals have
experiment to determine the validity of the kit is necessary. completely dissolved. The 1× wash solution is stable for 2 weeks at
4) Owing to the possibility of mismatching between antigen from other 2-8°C.
resource and antibody used in our kits (e.g., antibody targets 17) Do not dilute the other components which are ready- to-use.
conformational epitope rather than linear epitope), some native or
recombinant proteins from other manufacturers may not be 8. ASSAY PROCEDURE
recognized by our products.
5) Influenced by the factors including cell viability, cell number and Please read Reagents Preparation before starting assay procedure. It is
also sampling time, samples from cell culture supernatant may not be recommended that all Standards and Samples be added in duplicate to the
detected by the kit. microtiter plate.
6) Fresh samples without long time storage are recommended for the
test. Otherwise, protein degradation and denaturalization may occur 1) Secure the desired numbers of coated wells in the holder then add
in those samples and finally lead to wrong results. 100 µL of Standards or Samples to the appropriate well in the
antibody pre-coated Microtiter Plate. Add 100 µL of PBS (pH
7. REAGENTS PREPARATION 7.0-7.2) in the blank control well.
2) Dispense 10 µL of Balance Solution into 100 µL specimens, mix
14) Bring all kit components and samples to room temperature (20-25 well. (NOTE: This step is required when the sample is cell culture
°C) before use. supernatants, body fluid and tissue homogenate; if the sample is
15) Samples - Please predict the concentration before assaying. If serum or plasma, then this step should be skipped.)
concentrations are unknown or not within the detection range, a 3) Add 50 µL of Conjugate to each well (NOT blank control well). Mix
preliminary experiment is recommended to determine the optimal well. Mixing well in this step is important. Cover and incubate the
plate for 1 hour at 37°C.

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FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.