Sample Types Validated:Serum, blood plasma,Saliva, Urine, and other related tissue Liquid.
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Please carefully read this instruction before using. This ELISA kit
is based on the principle of double-antibody sandwich technique to
detect Human Vitamin D3(VD3). Be used only for research purposes,
not be used for medical diagnosis.
This kit is used to assay the Vitamin D3(VD3)in the sample of
Human’s serum, blood plasma, and other related tissue Liquid.
The kit uses a double-antibody sandwich enzyme-linked immunosorbent
assay (ELISA) to assay the level of Human Vitamin D3(VD3)in
samples. Add Vitamin D3(VD3) to monoclonal antibody Enzyme well
which is pre-coated with Human Vitamin D3(VD3)monoclonal antibody,
incubation; then, add Vitamin D3(VD3)antibodies labeled with
biotin, and combined with Streptavidin-HRP to form immune complex;
then carry out incubation and washing again to remove the
uncombined enzyme. Then add Chromogen Solution A, B, the color of
the liquid changes into the blue, And at the effect of acid, the
color finally becomes yellow. The chroma of color and the
concentration of the Human Substance Vitamin D3(VD3)of sample were
Materials supplied in the Test Kit
Chromogen Solution A
Chromogen Solution B
Str- HRP-Conjugate Reagent
Closure plate membrane
Materials required but not supplied
1. 37 ℃ incubator 2. Standard Enzyme reader
3. Precision pipettes and Disposable pipette tips 4. Distilled
5. Disposable tubes for sample dilution 6. Absorbent paper
1. Beening taken out from the 2-8℃ environment, the kit should be
balanced 30 minutes in the ambient temperature then use. If the
Coated plates of Enzyme haven’t been used up after opened, the
remaining plates should be stored in Sealed bag.
2. For each step, add Sample with sample injector which should be
calibrated frequently, in order to avoid unnecessary experimental
3. he operation shall be carried out accordance to the instructions
strictly. And test results must be based on the readings of the
4. In order to avoid cross-contamination, it is forBIDden to re-use
the suction head and seal plate membrane in your hands.
5. All samples, washing buffer and each kind of reject should
according to infective material process.
6. The idle agents shall be put up or covered. Do not use reagent
with different batches. And use them before expired date.
7. The substrate B is light-sensitive. Prolonged exposure to light
Manually washing method: shake away the remain liquid in the enzyme plates; place some
bibulous papers on the test-bed, and flap the plates on the upside
down strongly. Inject at least 0.35ml after-dilution washing
solution into the well, and marinate 1~2 minutes. Repeat this
process according to your requirements.
Automatic washing method: if there is automatic washing machine, it should only be used in
the test when you are quite familiar with its function and
Intra-assay Precision (Precision within an assay): 3 samples with
low, middle and high level Human VD3 were tested 20 times on one
Inter-assay Precision (Precision between assays): 3 samples with
low, middle and high level Human VD3 were tested on 3 different
plates, 8 replicates in each plate.
CV(%) = SD/meanX100
1. Can’t detect the sample which contain NaN3, because NaN3
inhibits HRP active.
2. extract as soon as possible after Specimen collection,and
according to the relevant literature, and should be experiment as
soon as possible after the extraction. If it can’t, specimen can be
kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.
3. serum- coagulation at room temperature 10-20 mins,centrifugation
20-min at the speed of 2000-3000 r.p.m. remove supernatant, If
precipitation appeared, Centrifugal again.
4.plasma-use suited EDTA or citrate plasma as an anticoagulant,mix
10-20 mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m.
remove supernatant, If precipitation appeared, Centrifugal again.
5. Urine-collect sue a sterile container, centrifugation 20-min at
the speed of 2000-3000 r.p.m. remove supernatant, If precipitation
appeared, Centrifugal again. The Operation ofHydrothorax and
cerebrospinal fluid Reference to it.
6.cell culture supernatant-detect secretory components, collect sue
a sterile container, centrifugation 20-min at the speed of
2000-3000 r.p.m. remove supernatant,detect the composition of
cells, Dilut cell suspension with PBS(PH7.2-7.4), Cell
concentration reached 1 million / ml, repeated freeze-thaw cycles,
damage cells and release of intracellular components,
centrifugation 20-min at the speed of 2000-3000 r.p.m. remove
supernatant, If precipitation appeared, Centrifugal again.
7.Tissue samples- After cutting samples, check the weight,add
PBS(PH7.2-7.4), Rapidly frozen with liquid nitrogen, maintain
samples at 2-8℃ after melting,add PBS(PH7.4), Homogenized by hand
or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m.
Contact Rebecca Yan