CN106932563B - A kind of preparation method of the ELISA standard dilutions based on serum - Google Patents
A kind of preparation method of the ELISA standard dilutions based on serum Download PDFInfo
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- CN106932563B CN106932563B CN201710039375.2A CN201710039375A CN106932563B CN 106932563 B CN106932563 B CN 106932563B CN 201710039375 A CN201710039375 A CN 201710039375A CN 106932563 B CN106932563 B CN 106932563B
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Abstract
The preparation method of the invention discloses a kind of ELISA standard dilutions based on serum, include the following steps, a. several animal blood serums of buffer are used, serum-concentration is 5%-95%, concentration gradient is 5%, and target protein is added in the animal blood serum of each concentration, takes the human serum sample of several health, target protein is added, is approached in target protein concentration and animal blood serum after preparation；B. conventional ELISA experimental implementation is done to the animal blood serum and human serum sample of each concentration after addition target protein；C. OD curve is made：Excel table horizontal axis is animal blood serum concentration, and the longitudinal axis is that OD value makes OD curve；D. the animal blood serum concentration intersected with human serum sample OD average value is determined, i.e., reaction character of the target protein in ELISA experiment is close with the animal blood serum of concentration shown in the intersection point in human serum；E. recovery testu is done using the animal blood serum of gained concentration in d, confirms that the rate of recovery whether in 80%-120%, if any deviation, finely tunes the animal blood serum concentration up and down.
Description
Technical field
The present invention relates to Soluble target albumen accurate quantitative analysis technical field, more particularly, to a kind of based on serum
The preparation method of ELISA standard dilutions.
Background technique
ELISA (enzyme-linked immunosorbent assay) is a kind of method for carrying out Soluble target albumen accurate quantitative analysis in sample.
The function of the core component standard dilutions of ELISA kit is ensuring that the accuracy of ELISA kit.Theoretically standard
The component of product dilution should be but the practical feelings except identical with the component of sample to be examined without other components in addition to target protein
Condition is that this is impossible, the especially serum, plasma sample of people.
Standard dilutions on textbook are the PBS-T solution containing 0.5%BSA, this solution for cells and supernatant,
The application effect of the sample type of the low protein concns such as tissue grinder liquid, Tissue lysates is also possible, and mark-on reclaims are usual
Between 80-120%.But for the sample of serum, the such high protein concentration complicated components of blood plasma, mark-on reclaims only have 20-
30%, it is to be grossly inaccurate that the result obtained in this way, which differs too big with actual concentrations,.Also the multiple calcemia slurry of user makees sample
This dilution, effect be it is quite a lot of, problem is potential infectiousness, limited amount, expensive, and there are also forbidding manufacture for law.
Summary of the invention
To solve deficiency in the prior art, the object of the present invention is to provide a kind of, and the ELISA standard items based on serum are dilute
Release the preparation method of liquid, it is intended to solve existing common standard product dilution to the sample mark-on reclaims of high protein concentration complicated components
Effect is poor, and the preferable standard dilutions of recovering effect are expensive, and potential problems are serious.
To achieve the above object, the technical scheme is that：A kind of system of the ELISA standard dilutions based on serum
Preparation Method includes the following steps,
A. several animal blood serums of buffer, serum-concentration 5%-95%, concentration gradient 5%, each dense are used
Target protein is added in the animal blood serum of degree, takes the human serum sample of several health, target protein, target protein after preparation is added
It is approached in concentration and animal blood serum；The pH of the buffer is 7.2~7.4；
B. the animal blood serum to each concentration after addition target protein and human serum sample do conventional ELISA experiment and grasp
Make；
C. OD curve is made：Excel table horizontal axis is animal blood serum concentration, and the longitudinal axis is that OD value makes OD curve；
D. the animal blood serum concentration intersected with human serum sample OD average value is determined, i.e., target protein exists in human serum
Reaction character in ELISA experiment and concentration shown in the intersection point of human serum sample OD average value and animal blood serum mark-on OD curve
Animal blood serum it is close；
E. do recovery testu using the animal blood serum of gained concentration in d, the confirmation rate of recovery whether in 80%-120%,
If any deviation, the animal blood serum concentration is finely tuned up and down.
Preferably, destination protein concentration is 200pg/ml in the animal blood serum.
In order to eliminate due to reagent is impure or reagent interference etc. caused by error, it is further comprising the steps of, in step a
Each concentration animal blood serum and health human serum do blank sample-adding, data background of the data obtained as step d.
In order to facilitate acquiring and reducing cost, the animal blood serum is using in lowlenthal serum, rabbit anteserum or fetal calf serum
It is a kind of.
Preferably, the buffer is 0.5%Tween 20PBS.
Preferably, the buffer is 0.5%Tween 20TBS-T.
Beneficial effects of the present invention：Mark-on reclaims effect is good, and price is lower, easy to make.
Detailed description of the invention
Fig. 1 is the OD curve graph of the embodiment of the present invention 1.
Specific embodiment
Embodiment 1, a kind of preparation method of the ELISA standard dilutions based on serum,
A. lowlenthal serum, concentration gradient 5% are prepared with 0.5%Tween 20PBS series, concentration fraction is respectively 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, the H-IL-6 standard protein that 5ul concentration is 4000pg/ml is added in 90%, 95%, each lowlenthal serum concentration 95ul,
The final concentration of 200pg/ml of standard protein；4 Healthy Human Serums are taken, the H-IL-6 that 5ul concentration is 4000pg/ml is added in 95ul
(concentration of general health human serum H-IL-6 is in 20pg/ml hereinafter, the standard protein concentration of serum is about after mark-on for standard protein
200pg/ml, if encounter H-IL-6 expression it is higher, reject its data)；
B. by each concentration lowlenthal serum after mark-on and the human serum sample after mark-on, pre-coated H-IL-6 enzyme mark is added
The hole plate 50ul/ adds the H-IL-6 detection antibody in the hole 50ul/, and 300 revs/min of room temperature are incubated for for oscillation 2 hours, is washed out
Liquid washs 5 times, and the horseradish peroxidase in the hole 100ul/ is added, and subsequent cleaning solution washs 5 times, and the hole 100ul/ TMB colour developing is added
Substrate develops the color 10 minutes, and the sulfuric acid of 2M terminates reaction；Gained microplate reader Detection wavelength 450nm, reference wavelength 630nm read OD
Value；
C. OD curve is made；
D. by OD curve graph it is found that the OD of the mean OD value of human serum sample 200pg/ml mark-on and lowlenthal serum mark-on is bent
Line intersects at 60%, i.e., reaction character of the H-IL-6 in ELISA experiment is close with 60% concentration lowlenthal serum in human serum.
E. using 60% concentration lowlenthal serum as the standard dilutions of H-IL-6,10 people's serum samples do mark-on reclaims
Experiment, rate of recovery range 99%-115%, average recovery rate 106%.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modification, equivalent replacement or improvement etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (6)
1. a kind of preparation method of the ELISA standard dilutions based on serum, which is characterized in that include the following steps,
A. several animal blood serums of buffer, serum-concentration 5%-95%, concentration gradient 5%, in each concentration are used
Target protein is added in animal blood serum, takes the human serum sample of several health, target protein, target protein concentration after preparation is added
It is approached with animal blood serum；The pH of the buffer is 7.2~7.4；
B. conventional ELISA experimental implementation is done to the animal blood serum and human serum sample of each concentration after addition target protein；
C. OD curve is made：Excel table horizontal axis is animal blood serum concentration, and the longitudinal axis is that OD value makes OD curve；
D. the animal blood serum concentration intersected with human serum sample OD average value is determined, i.e., target protein is in ELISA reality in human serum
Reaction character and the animal blood of concentration shown in the intersection point of human serum sample OD average value and animal blood serum mark-on OD curve in testing
It is clear close；
E. do recovery testu using the animal blood serum of gained concentration in d, the confirmation rate of recovery whether in 80%-120%, if any
Deviation finely tunes the animal blood serum concentration up and down.
2. a kind of preparation method of the ELISA standard dilutions based on serum according to claim 1, which is characterized in that
Target protein concentration is 200pg/ml in the animal blood serum.
3. a kind of preparation method of the ELISA standard dilutions based on serum according to claim 1, which is characterized in that
Further comprising the steps of, the human serum of animal blood serum and health to each concentration in step a does blank sample-adding, the data obtained
Data background as step d.
4. a kind of preparation method of the ELISA standard dilutions based on serum according to claim 1, which is characterized in that
The animal blood serum is using one of lowlenthal serum, rabbit anteserum or fetal calf serum.
5. a kind of preparation method of the ELISA standard dilutions based on serum according to claim 1, which is characterized in that
The buffer is 20 PBS of 0.5%Tween.
6. a kind of preparation method of the ELISA standard dilutions based on serum according to claim 1, which is characterized in that
The buffer is 20 TBS-T of 0.5%Tween.
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| CN109060972B (en) * | 2018-05-16 | 2021-09-24 | 苏州药明泽康生物科技有限公司 | Application of rabbit blood in preparing human disease in-vitro diagnosis kit |
| CN112415211A (en) * | 2020-10-23 | 2021-02-26 | 杭州联科生物技术股份有限公司 | Simple preparation method of standard product diluent |
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Denomination of invention: A preparation method of serum based ELISA standard dilution Effective date of registration: 20211206 Granted publication date: 20181123 Pledgee: Hangzhou High-tech Financing Guarantee Co.,Ltd. Pledgor: MULTI SCIENCES(LIANKE) BIOTECHNOLOGY CORPORATE LTD. Registration number: Y2021330002461 |
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Date of cancellation: 20221027 Granted publication date: 20181123 Pledgee: Hangzhou High-tech Financing Guarantee Co.,Ltd. Pledgor: MULTI SCIENCES(LIANKE) BIOTECHNOLOGY CORPORATE LTD. Registration number: Y2021330002461 |
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