Kit for detecting canine progesterone by enzyme-linked immunosorbent assay (ELISA) and preparation method thereof

A technology of enzyme-linked immunosorbent adsorption and detection kit, which is applied in biological testing, measuring devices, material inspection products, etc. Accurate quantitative analysis and other issues, to achieve the effect that is conducive to detection sensitivity and specificity, broad market application prospects, and favorable detection

Pending Publication Date: 2021-05-07
爱若维生物科技(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The content of canine progesterone presents regularity in different stages of estrus. Clinical detection methods include instrumental methods: chemiluminescence instruments, liquid chromatography mass spectrometry and high-efficiency chromatography. Higher requirements require the use of supporting reagents
Also based on immunochromatography detection, the prepared colloidal gold detection test strip can quickly detect the results, but the sensitivity of this method is insufficient, and it cannot be accurately quantitatively analyzed
The prepared quantum dot detection test strip has high sensitivity and can quickly detect the results, but the detection throughput is low, and only one can be detected at a time, which is expensive for large-scale breeding units

Method used

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  • Kit for detecting canine progesterone by enzyme-linked immunosorbent assay (ELISA) and preparation method thereof
  • Kit for detecting canine progesterone by enzyme-linked immunosorbent assay (ELISA) and preparation method thereof
  • Kit for detecting canine progesterone by enzyme-linked immunosorbent assay (ELISA) and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Preparation of pre-coated microwell plate, sample diluent, chromogenic reagent, washing solution, standard substance, and termination reagent

[0042] The material of the microwell plate is NUNC plate with high binding capacity, (article number: 400011-5), add canine progesterone antigen 100ug / well into the NUNC plate, coat it overnight at 4, absorb the antigen, add 5% milk to block After 30 minutes, discard and dry overnight at room temperature under vacuum; store in a vacuum-sealed aluminum foil bag with desiccant.

[0043] Sample diluent: 5% milk with 0.01% Procline200.

[0044] Chromogenic reagent: ABTS (KPL).

[0045] Washing solution: 0.01 MPBS buffer.

[0046] Standard product: progesterone content 0.1ug antibody protectant solution (prepared in the laboratory, LOT: 20200813).

[0047] Termination reagent: 1.25% NaF solution.

Embodiment 2

[0048] Embodiment 2 Preparation method of enzyme-labeled secondary antibody

[0049]Enzyme-labeled secondary antibody: HRP enzyme-labeled canine progesterone antibody (laboratory preparation: LOT: 20201029), labeling process: 1mg HRP was dissolved in 40ul of 0.25mol / l PBS, added 10ul of 25% glutaraldehyde, and incubated at 37 degrees for 2h Then add 0.2ml of cold absolute ethanol, centrifuge at 2500rpm for 15min, discard the supernatant, use 1ml of 0.05M carbonate buffer solution for precipitation, add 2mg / ml canine progesterone antibody, mix well, refrigerate overnight, add KH 2 PO 4 0.5ml, mix well, aliquot and freeze at -20°C.

Embodiment 3

[0050] Example 3 Canine progesterone ELISA detection method one

[0051] (1) Dilute canine serum or plasma 50 times with PBS and add it to the microwell plate, 100ul / well, and add the standard substance at the same time, the standard substance is diluted 6 gradients, the canine progesterone in the serum or plasma will be mixed with the microwell plate Antigen-specific binding, incubate at 37°C for 30min, then wash 3 times, and blot dry;

[0052] (2) Add enzyme-labeled secondary antibody 1ug / ul, 100ul / well to react, incubate at 37°C for 30min, then wash 3 times, and dry;

[0053] (3) Add ABTS, develop color at room temperature for 30 minutes, and read with a microplate reader, with wavelengths of 405 nm and 492 nm, and read within 2 minutes;

[0054] (4) The obtained result sample OD=△405-△492, make a standard curve, and calculate the canine progesterone content according to the standard curve and the test results of the sample.

[0055] The assay structure is shown in Table ...

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Abstract

The invention relates to a kit for detecting canine progesterone by enzyme-linked immunosorbent assay (ELISA), which realizes detection of multiple samples in a short time through specific selection and matching of a pre-coated microwell plate and an enzyme-labeled secondary antibody, can perform high-throughput, high-sensitivity and low-cost detection on the content of canine progesterone, and meets the requirements of clinical detection.

Description

technical field [0001] The invention belongs to the field of in vitro diagnosis of animal medical devices, and relates to a canine progesterone detection kit, in particular to a canine progesterone enzyme-linked immunosorbent assay (ELISA) detection kit. Background technique [0002] Canine progesterone is a steroid hormone secreted by the corpus luteum. It plays an important role in the reproductive activities of female dogs, such as ovulation, maintenance of pregnancy, initiation of childbirth, and mammary gland development. Clinical detection of canine progesterone can determine the best mating period for dogs. By monitoring the decline of progesterone value, it can determine the time of delivery, and also determine whether progesterone supplementation is needed during pregnancy to increase the conception rate. Significant, it is of great help to dog production and breeding units. [0003] The content of canine progesterone presents regularity in different stages of est...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/74G01N33/543G01N33/535
CPCG01N33/743G01N33/54306G01N33/535
Inventor 王蓉蓉冯小龙孙云雷张施羽钱翀
Owner 爱若维生物科技(苏州)有限公司
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