Dairy cow ovulation colloidal gold test strip
Dairy cow ovulation colloidal gold test strip specification
For Professional and In Vitro Diagnostic use only
Immunoassay for Qualitative Detection of Ovulation in dairy Cow
【INTENDED USE】
This test is an immune-chromatographic one step assay designed for in vitro qualitative determination of cow luteinizing hormone (LH) in milk to predict time of ovulation.
【SUMMARY AND EXPLANATION】
LH (Luteinizing Hormone) is a kind of glycoprotein hormone, just like FSH (Follicle-Stimulating Hormone), it’s one of the main factors which induced ovulation. LH composed by two subunits αand β，among themαis the hydrophilic chain andβis the hydrophobic chain. The αof LH is very similar to the αof HCG,FSH, TSH, they have the same antigenic determinant，but their respective hormone biological characteristics is determined by the βsubunit. The αand βof LH are combined through non covalent bond, so, the combination is unstable and easy to fracture. That is to say the two subunits of LH are combined by electrostatic adsorption and so on. The composition of the dairy cow milk is too complication which not only has water-soluble matter, but also has a lot of fat-soluble substances, such like milk fat. Theβof LH is easier to dissolved in milk fat，so, it is easier to catch the peak of LH by test the βsubunits in milk fat. In female mammals, LH is cooperating with FSH to aid the follicle growing maturely and motivate the ovulation. In addition, it enhances the blood flow. Ovulation is the process in which the ovary releases the ovum, a nd it’s the key event in ov ary period. The ovulation of mammals is a complex process involved with many genes and steps. A lot of research indicates that ovulation is regulated by hormone and partial active substance of follicle. Several or more hours before the ovulation of mammals, secretion of LH would rise in response to female hormone positive regulation, and LH surge appears. After the LH surge, the wall of the enlarged follicle ruptures at ovulation and the mature ovum is extruded. After ovulation, LH returns to its base line level with the concomitant increase of progesterone level to initiate luteal phase. Before ovulation, the LH in blood, milk and urine would rise gradually, and approximately 12-23 hr after the LH surge, the ovulation will occur.
In view of the characteristic variation of LH, rapid and sensitive measurement of LH is important to diagnose and manage of pregnancy for female mammals. We can predict the ovulation time by tracing and detection of the LH surge.
【PRINCIPLE OF THE METHOD】
This test is a qualitative, two-side sandwich colloidal gold immune-chromatography assay for the determination of cow Luteinizing hormone (cLH) in milk. The nitrocellulose membrane is pre-coated with goat anti-cLH monoclonal antibody on the test band region and goat anti-mouse polyclonal antibody on the control band region.When the concentration of LH in the sample reaches a certain level, the LH in the milk would bind to the gold labeled anti-LH monoclonal antibody (Ⅰ)forming a complex of Ag-Ab-Au. Due to capillary chromatography, this complex moves along
the membrane band, and binds to the anti-LH monoclonal antibody (Ⅱ) forming a two-side sandwich colloidal immune complex. A color band (T band) will appear in the T area; the surplus gold labeled anti-LH monoclonal antibody (Ⅰ)would continue moving, and a visible color band (C band) will appear at the control area since it comes from mouse. (Result A)If both T band and C band are visible and the T band is darker or equal to the C band, the test result is Positive, which means the LH surge is coming, and the ovulation would happen within the next 12-23hr; (Result B)when the LH concentration is lower than 10mIU/ml, only one pink band appears in the control area, or the test line band is lighter than the control band, which demonstrates that the result is Negative and no LH surge appears.
【CLINICAL TRIALS】
The ovulation of dairy cow is unlike humans, most of cows have rutting performance: ①The change of behavior: the rutting cows are excited, often sing and walking around on the playground, chasing and climbing across the other cows, or accepting climbed by other cows, hance,frequent urination, lifting or shaking the tail, losing of appetite.②The change of reproductive organs: vulva is relaxation,swelling, mucosal hyperemia, flush, the cervix opening, endocrine mucus discharge from the vulva, mucus is transparent and linear. The different traditional methods of insemination were designed based on the behavior above described.
(1)The capture rate of LH peak is influenced by many factors,associated with human factor
closely, especially, rutting in the night, the LH peak was missed often due to lacking of observation. So, the LH peak capture rate can get higher up to 90%, generally, it was about 50-78%. The LH peak capture rate was 66.5% in clinical trial of 709 cows; among them the LH peak capture rate was 20.8% after rutting 0-6 hr; it was 17.9% after 6-12hr and 14.1% after 12-24hr.
(2)Ovulation test strip contrast with B ultrasound
The LH peaks of 31 estrus cows tested with the ovulation strips contrast with the changes of ovarian follicle observed with B ultrasound. When LH peak appeared, the ovarian follicle was in the state of high filling detected with B ultrasound, and, generally, the ovulation happened in 12 hr. When rupture and disappeared of follicles were observed with B ultrasound, the LH peak was negative tested with strip and the total coincidence rate got up to 93.3%.
【SCOPE AND APPLICATION】
（1）It is suitable for prediction cow ovulation and mating timely, which can improve the conception rate. According to the differences of physical, chemical and biological between the sperm X and Y, such as characters of Density, size, weight, shape, energy, surface charge, and the motility of sperm Y is stronger; survival time is shorter in the vagina, so, if the fertilizing was after the ovulating, the sperm Y could combine with egg and form male embryos firstly. On the contrary，if the fertilizing was before ovulating ,because of the activity of sperm Y was weakening in the vagina, the sperm X could combine with egg to form female embryos.To confirm the exact time of ovulation and according to the characteristics of sperm X/Y, the sex selection and eugenics could be performed.Because cows ovulation mainly occurs in10-20 hr after rutting , and the survival time of sperm Y in female reproductive tract is about 8-10hr, if the LH peak could be detected in the early stages of rutting, and to do insemination
immediately, then most sperm X combined with egg ,so, the female proportion will increase in offspring. The reason is the energy of sperm Y costs a lot and it’s enzymes has been disabled mostly, result in sperm Y life was shorten. So that, the sperm Y lost the ability of competing with sperm X. 【SPECIFICITY 】
There were not cross reaction observed with standard substance of 500ng/ml Follicle Stimulating Hormone (FSH), 500ng/ml estrodiol, 500ng/ml prolactin, 20μg/m l acetaminophen, 20μg/m l acetylsalicylic acid, 20μg/m l tetracycline and 0.01nol/ml pH=7.3 PBS.
【S
AMPLING TIME 】
The sample was collected from the rutting cows, and used to test the LH level with colloidal gold test strip.
【THE PRETREAMENT METHOD OF THE SAMPLE 】
The sample was on standing for 30-60 min or centrifuged to collect the cream from milk which was used to test. When the test performed, taking one part of the cream first, then adding one part of anhydrous ethanol for helping to dissolve, and adding two parts of water diluted the sample.
【ASSAY PROCEDURE 】
Test strip:
1. Remove the test strip from the foil pouch.
2. Put the “MAX” end into the sample (the surface of the sample should not exceed the MAX line). Due to the function
of capillary, the sample dissolves the solid antibody labeled with colloidal gold and moves forward to the other end. When the liquid moves to the 2 / 3 of the result window or to the end, you can take out the strip. One or two red lines will appear in the result window.
3. Observe the result in 10 min. Do not exceed 20 min.
Negative: If only the control band (C) appears, or the color intensity of the Test line is less than that of Control line ,it is suggested that the diary cow is not pregnant.
Suspicious: If the color intensity of the test band (T) is less than that of control band
(C), the specimen should be double-checked in another day until LH surge appears. Invalid: When neither test band nor control band appears on the membrane, the test should be invalid, probably, since improper test procedure or deterioration of reagents occurred. It is suggested that the test would be repeated.
Cassette:
1. Remove the test device and milk dropper from its foil wrapper by tearing along the slice.
2. Using the milk dropper, withdraw the milk sample from the specimen cup and slowly dispense 2～3drops into the circular sample well. The sample liquid moves slowly to the other end due to the capillarity. You will see one or two red lines in the result window.
3. Read results in 10 minutes. Do not exceed 20 min.
Positive: If the colour of test band (T) was more intensive than or equivalent to
the control band (C), the LH peak already appeared, and ovulation
would come soon.
Negative: If only the control band (C) appeared, that mean the LH peak did not
happen.
Suspicious: If the color intensity of the test band (T) was light than the control band
(C), then the specimen should be double-checked in another day until LH peak appeared.
Invalid: When neither test band nor control band appeared on the strip, the test
should be invalid, which due to, probably, the improper test procedure or the deterioration of reagents occurred. It is suggested that the test would be repeated.
【Sensitivity; Accuracy rate; Precision 】
The minimum detectable amount: 10miu; accuracy rating ＞90%; Precision ＞90%.
【PRECAUTION 】
1. Do not reuse the test devices.
Suspicious
2.Once opened, the test device should be used immediately. Prolonged exposure to
atmosphere will lead to deterioration.
3.While doing the detection, make sure that the surface of sample should not exceed
the “MAX” line of strip.
4.Ensure the cream is absorbed accurate when sample processing.
5.Pay attention to observation on cow physiological changes timely, otherwise, the LH peak
of rutting was missed in the night.
6.To use the test kit in the period of validity.
【SPECIFICATIONS】
For strip: 1 Strip/pouch×100pouches/box;
For Cassette: 1share/cassette×40 cassettes /box;
【THE PERIOD OF V ALIDITY】
The period of the validity is 24 months.
【STAORAGY AND STABILITY】
Stored at 4-25℃ in dry space, keep away from light, and do not be frozen. 【Manufacturer】
W.H.P.M. Bioresearch & Technology Co. Ltd
Address: No.2 Zhongxin Street, LouZiZhuang, Jinzhanxiang, Chaoyang District, Beijing P.R. China, 100018
Post code: 100018
Tel: 0086-010-********
Fax: 0086-010-********
Mobile phone: 139******** 136********
E-mail:jichengwan@https://www.sodocs.net/doc/1f15125521.html,