Span Biotech Ltd.

INTENDED USE

This test is an immunochromatographic 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 one kind of glycoprotein hormone. Just like FSH (Follicle-Stimulating Hormone), it’s one of the main factors motivating the ovulation. 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 releasing the ovum, and it’s the key event in ovary 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 hours after the LH surge, the ovulation will occur.

In view of the characteristic variation of LH, rapid and sensitive measurement of LH is an important tool in the diagnosis and management of infertility in female mammals. We can forecast the happening of ovulation by tracing and detection of the LH surge.

PRINCIPLE

This test is a qualitative, two-site sandwich colloidal 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 binds to the gold labeled anti-LH monoclonal antibody Ⅰforming 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-site sandwich colloidal immune complex. A color band (T band) will appear in the T area; the surplus gold labeled anti-LH monoclonal antibody Ⅰwould 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 12-23hours; (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.

REAGENTS AND MATERIALS

Each test device is individually sealed in a foil pouch.

Test Device:

1.Containing test dipcard with captured anti-cLH antibody coated membrane and colored anti-cLH monoclonal antibody pad.

2.Desiccant

Materials Required But Not Provided

Timer, specimen container.

STAORAGY AND STABILITY

Stored at 4-25℃ in dry space, avoiding light, and can not be frozen. The test strip is stable until the date imprinted on the pouch label (18 months).

PRECAUTION

1.Read directions for use carefully before performing this test.

2.Do not use test kit beyond expiration date.

3.Do not reuse the test devices.

4.Discard it into the dustbin after single use.

5.Once opened, the test device should be used immediately. Prolonged exposure to atmosphere will lead to deterioration.

6.While doing the detection, make sure that the surface of sample should not exceed the “MAX” line of strip.

ADAPTIVE RANGE

The test strip is a rapid one step assay for the detection of dairy cow early ovulation, inseminating timely, enhancing insemination rate, and making the sex choice of cow according to the characteristics of X, Y sperms.

SPECIMEN COLLECTION AND PREPARATION

This colloidal gold test strip is designed for the qualitative determination of LH in cow milk. The sample cow milk should stand still for 30min-1hr or centrifuge for 5min at 300rpm so that the cream could be collected for testing. 95% ethanol should be added (v/v = 1:1) to help solving the cream, then equal volume of water should be added to the solved cream for dilution.

ASSAY PROCEDURE 

1.Remove the midstream test 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.

3.Observe the result from the window within 10min, but no longer than 20 min

Cassette:

1.Remove the test device from its foil wrapper by tearing along the slice.

2.Using the urine dropper, withdraw the urine sample from the specimen cup and slowly dispense 3 drops into the circular sample well. When the sample liquid moves slowly to the other end due to the capillarity, it will dissolve the solid gold labeled antibody and brought it ahead in the mean time.

3.Read results within 10 min. Do not read after 20 min.

INTERPRETATION OF RESULTS

Positive: If the test band (T) is more intensive than or equivalent to that of control band (C), the specimen is likely at or around the surge level, and ovulation would come soon.

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 nor control band appears on the membrane, the test should be voided since improper test procedure or deterioration of reagents probably occurred. It is recommended that the test be repeated.

Cassette:

Positive: If the test band (T) is more intensive than or equivalent to that of control band (C), the specimen is likely at or around the surge level, and ovulation would come soon.

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.

Invalid: When neither test nor control band appears on the membrane, the test should be voided since improper test procedure or deterioration of reagents probably occurred. It is recommended that the test be repeated.

PERFORMANCE CHARACTERISTICS

1.SENSITIVITY

This test has been designed to produce a definitive color band at test region when tested with 10mIU/mL or higher of LH at room temperature.

2. SPECIFICITY

Specificity of this test was determined from cross reaction studies with 500ng/mL Follicle Stimulating Hormone (FSH), 500ng/mL estrodiol, 500ng/mL prolactin, 20μg/mL acetaminophen, 20μg/mL acetylsalicylic acid, 20μg/mL tetracycline and 0.01nol/mL PH=7.3 PBS. No cross reactivity was observed.

3.   ACCURACY

In laboratory studies, this test has been proven to be 90% accurate.

SPECIFICATIONS

For strip:1 Strip/pouch╳100puches/box;

For cassette:1 piece/cassette╳40cassettes/box