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Quantitative detection reagent for activity of semen lactate dehydrogenase X isozyme

A lactate dehydrogenase and quantitative detection technology is applied in the field of quantitative detection of semen lactate dehydrogenase X isoenzyme activity, which can solve the problems of narrow detection range, insufficient sensitivity, poor substrate stability, etc., and achieve accurate and reliable results and detection. Wide range and good stability

Inactive Publication Date: 2007-01-24
深圳华康生物医学工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing semen LDH-X activity quantitative detection reagent (such as: substrate adopts phosphate buffered saline), has the following deficiencies: 1. The detection range is narrow and cannot cover normal and abnormal pathological ranges
2. It is difficult to avoid the interference of metal ions on the detection of isozymes
3. The stability of the substrate is poor and the storage period is short, so it is not suitable for use as a kit
[0004] The determination of human sperm LDH-X mainly includes polyacrylamide electrophoresis and spectrophotometry. The former is complicated to operate and not sensitive enough, and it detects the amount of LDH-X rather than its activity.
The latter uses the rate method to detect the activity of LDH-X, which is simple to operate, but has the following disadvantages: 1. Due to the rapid change of data before and after the reaction, the measurement error is very large, which cannot meet the manual operation requirements of clinical detection reagents, and can only be used in Detected on an automatic biochemical analyzer; 2. The reaction time cannot be precisely controlled; 3. Since there is no temperature control device on the spectrophotometer, the temperature cannot be controlled (required reaction temperature is 37°C)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1, a kind of semen lactate dehydrogenase X isozyme (LDH-X) activity quantitative detection reagent, comprises: (1) substrate A (lyophilized powder): β-NADH content is the Tris of 0.05~0.5% -EDTA-Na 2 Buffer or buffer freeze-dried powder; (2) Substrate B (lyophilized powder): Tris-EDTA-Na with a carbon chain greater than 5 2-keto acid content of 0.5-5% 2 Buffer or buffer lyophilized powder, 2-ketoacids with more than 5 carbon chains, preferably 2-ketocaproic acid; (3) Stopping solution: containing sodium dodecyl sulfate (SDS) 3-10% Ionic water solution.

[0018] When the detected semen lactate dehydrogenase X isoenzyme activity is low, it is advisable to choose substrate A with low concentration of β-NADH content and substrate B with low concentration of 2-keto acid with more than 5 carbon chains. The amount of substance A and substrate B is reduced accordingly. When the detected semen lactate dehydrogenase X isozyme activity is high, it is advisable to cho...

Embodiment 2

[0032] Embodiment two, a kind of seminal fluid LDH-X activity quantitative detection reagent, comprises: (1) substrate A (lyophilized powder): β-NADH content is the Tris-EDTA-Na of 0.114% 2 Buffer or buffer lyophilized powder; (2) Substrate B (lyophilized powder): Tris-EDTA-Na with 2-ketocaproic acid content of 1.122% 2 Buffer or buffer lyophilized powder; (3) stop solution: deionized water containing 5% sodium dodecyl sulfate (SDS). Its preparation process comprises the steps of:

[0033] A. Tris-EDTA-Na 2 Buffer production process

[0034] 1), weigh 34.0 grams of Tris, EDTA-Na 2 10.4g, placed in a beaker. Add 900ml of deionized water, stir and dissolve in a 37°C water bath.

[0035] 2) Adjust the pH to 8.4 with 1mol / L HCl on a pH meter, transfer it to a volumetric flask, and dilute to 1000 ml with deionized water. Store at 4°C.

[0036] B, the manufacturing process of substrate A

[0037] 3) Weigh 1.140g of β-NADH, add appropriate amount of Tris-EDTA-Na 2 After the...

experiment example

[0045] Experimental example, specific steps for detecting human semen LDH-X with LDH-X quantitative detection reagent:

[0046] 1. Reagent preparation

[0047] Substrate A freeze-dried powder and substrate B freeze-dried powder were reconstituted with 16ml of distilled water.

[0048] 2. Sample preparation

[0049] 1. Seminal plasma: After the fresh semen is liquefied, quickly mix the specimen thoroughly, take part of the semen at 3000g and centrifuge for 15-20 minutes, and keep the seminal plasma for inspection.

[0050] 2. Semen: After the fresh semen is liquefied, quickly mix the specimen thoroughly, and count the sperm concentration of this specimen (M×10 6 / ml). After freezing and thawing once at -20°C, centrifuge at 3000g for 15-20min, and keep the seminal plasma for inspection.

[0051] 3. Speed ​​method

[0052] 1. Adding samples: add 750ul of substrate A solution and 750ul of substrate B solution into the same reaction tube. Pre-warm at 37°C for 5 minutes, add 1...

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Abstract

This invention publics an active quantitative determination reagent for sperm lactate dehydrogenase X isoenzyme (LDH-X) comprises substrate A, substrate B and terminating liquid. The substrate A contains Tris-EDTA-Na#-[2] cushioning liquid or freeze-drying powder of it with beta-NADH, and substrate B contains Tris-EDTA-Na#-[2] cushioning liquid or freeze-drying powder of it with Beta-NADH whose 2-ketoacidosis acid is that the carbonaceous chains are more than five. Through formulating substrate A substrate B, one can automatic detect LDH-X in the biochemical analytic instrument by method of velocity, and through confecting terminating liquid, one can handcraft detect LDH-X by method of two dots. It also public reagent case made adopting the above agent.

Description

technical field [0001] The invention relates to an in vitro diagnostic reagent, which is particularly suitable for the quantitative detection of semienzyme lactate dehydrogenase X isoenzyme (hereinafter referred to as LDH-X). Background technique [0002] LDH-X is mainly distributed in the cytoplasm of spermatocytes, spermatids, and spermatozoa, and the thick mitochondria. Its synthesis starts from the middle pachytene stage of primary spermatocytes until sperm formation, and its synthesis is regulated by androgen secretion. Impact. LDH-X provides sufficient energy for the movement of sperm in the reproductive tract. The measurement of semen LDH-X combined with the measurement of hormone levels can be used as an indicator for evaluating the state of the seminiferous epithelium; LDH-X is also a reliable indicator for evaluating the quality of semen. [0003] Existing semen LDH-X activity quantitative detection reagents (such as: phosphate buffered saline as a substrate) have...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/573G01N21/31
Inventor 傅剑华刘瑜胡家纯何林何小红
Owner 深圳华康生物医学工程有限公司
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