DESC:THE PATENTS ACT 1970

COMPLETE SPECIFICATION
(See section 10 and rule 13)

TITLE OF THE INVENTION

A novel point-of-care diagnostic kit for detection of semen samples in general sexual wellness and forensic examinations

APPLICANT

Human Biogenesis Private Limited
107, Mulgram, Uttarpara, P.O.: Chanduli
District- Purba Bardhaman, Pincode -713502, West Bengal, India.

The following specification particularly describes the invention and the manner in
which it is to be performed.

Field of the invention:
The current invention is designed to identify the traces of semen present either in vaginal swabs or on contaminated surfaces found upon the occurrence of unprotected sexual activities or during sexual assaults. This delivers dual importance to the kit in both medical and forensic examination grounds. The technology behind the development of this kit follows the concept of lateral flow immunoassay, immunology, molecular biology and colourimetry-based biochemistry.
Background of the Invention:
Unprotected sex can result in unwanted pregnancy and the current practice in India to avoid such unwanted pregnancy is to depend on contraceptive pills or birth control pills. Apart from delivering the targeted function, overconsumption of these pills can result in serious side effects including infertility. Semen is often found to be absent inside the vagina of the female sexual partner in cases of controlled ejaculation during intercourse. Therefore, consuming contraceptive pills becomes unnecessary in such events. However, due to the absence of a proper diagnostic device, it is difficult to attain surety about the presence of semen inside the vagina and in case of wrong analysis, it can lead to unwanted pregnancy. Hence, the need of the time is to have a handy, easy-to-use in vitro diagnostic (IVD) kit that can deliver the desired result at a point-of-care site, with accuracy, sensitivity and at a cheaper cost. Having such a kit saves female sexual partners from blind consumption of contraceptives and helps to avoid their unwanted side effects.
Besides the medical diagnostic field, this kit also has profound importance in the field of forensic science. Cases of sexual assaults like rape often involve the activity of forceful unprotected sex when the semen sample of the criminal is possible to be present in the body of the victim, or on the site of the assault. Investigation of such crimes by law enforcement and forensic officers often involves the examination of the presence of semen at the site of the crime because semen identification is the best positive evidence to confirm the incidence of sexual assault. The current invention is considered to be helpful in such kind of crime investigation since it offers a handy pocket-size portable device that is easy to carry to any crime scene, stable at room temperature and therefore avoids the need of cold storage or cold logistics, and highly specific and sensitive towards semen-specific reactants that enables to identify small amount of semen while at the same time will rule out false positive detection of other body fluids like vaginal secretion, urine or blood.
Existing forensic assay kits for semen detection apply the science of biochemical reaction involving prostatic acid phosphatase which is present in semen in high amounts and identification of spermatozoa through microscopic examination. However, the drawbacks of these kits are that endogenous vaginal acid phosphatase exists at such levels that its upper limit may overlap with the lower limit of the semen acid phosphatase. Hence, the possibility of the occurrence of false positive results is present. The present invention involves a dual detection method (engaging immunological and biochemical reactions) for semen identification and thus increases the chance of positive detection of semen samples while minimizing the events of false positive results. Also, being a handy point-of-care device, it is easier to carry to crime scenes, compared to the bulky microscopes.

Object of the Invention:
The object of the present invention is to offer society a handy, low-cost, user-friendly and low-maintenance point-of-care device with high specificity and sensitivity towards semen samples that can aid society and medical science by stopping unnecessary contraceptive consumption and reduce the associated health hazards.
The object of the present invention is also to help the government, law enforcement and forensic science in the identification of semen samples as crime evidence in cases of sexual assaults.
The object of the present invention is to develop a Lateral Flow Immunoassay-based in vitro diagnostic (IVD) kit.
Summary of the Invention:
The present invention consists of a plastic cassette comprising four types of fibre pads and membranes. These pads and membranes will contain the specific antibodies and chromogenic substrates. The antigens/reactants will be supplied through the semen samples under examination. The sample under examination will be collected by cotton swabs and will be extracted in the liquid buffer, all of which will be supplied with the kit. Sample extracted buffer will then be applied onto the sample application chamber of the kit cassette. If the sample contains semen antigens/reactants, then a positive coloured band will appear on the assigned test line regions on the test pad at the result displaying windows. A control line 6 will also appear to ensure a positive flow of the reactants over the applied reagents at the test lines. Noteworthy, the appearance of the control line 6 will be positive in both cases of the presence or absence of semen in the examined samples. Therefore, the appearance of two test lines will only confirm a positive reaction. In addition, the present invention will also allow simultaneous detection of semen acid phosphatase by the biochemical colourimetric reaction of converting colourless 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitrotetrazolium Blue (NBT) solution into intensified purple coloured products on the Acid Phosphatase (ACP) Test Strip 5.
Brief description of the drawings:
The foregoing summary, as well as the following detailed description of preferred embodiments, are better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings exemplary constructions of the invention; however, the invention is not limited to the specific methods and system disclosed. In the drawings:
Figure 1: Selection of nitrocellulose membrane using dipstick method to develop test lines. (a) FF80HP Plus, (b) FF80HP (c) FF120HP Plus Thick (d) FF120HP Plus (e) FF120HP (f) FF170HP Plus (g) FF170HP (h) FF170HP Plus Thick (i) Immunopore RP (j) Prima 40. The green highlighted ones showed positive result for test line development.
Figure 2: Selection of conjugate pad by dipstick method and development of control line 6 and test line 7. ST17 (left) and Fusion 5 (right) conjugate pads applied with 40 nm conjugate used in otherwise identical assembled strip.
Figure 3: Selection of gold nanoparticle conjugates. 20nm (left), 40nm (middle) and 80 nm (right) gold nanoparticles conjugated with capture antibody were applied to ST17 conjugate pads and 100ul of 1:20 extracted semen sample run by dipstick method.
Figure 4: Tests of assembled strips stored under different storage conditions i.e conjugate pad with (a) 10% sucrose, (b)15% sucrose and (c) 20% sucrose stored at 4oC for 15 days which are in contrast to (d) 10% sucrose in conjugate pad stored at room temperature for just 7 days.
Figure 5: Brown colour formation of alpha-naphthyl phosphate solution with Fast Blue BB indicator spotted on a Whatman grade 1 filter paper and left to dry for 1 h at room temperature.
Figure 6a: BCIP (left) and NBT+BCIP (right) spotted on Whatman grade 1 filter paper and 10µl of semen sample applied to the spot with pipette after 3 days storage at room temperature.
Figure 6b: BCIP (right) and NBT+BCIP (left) spotted on Whatman grade 1 filter paper in various concentrations and stored for 30 days at 40C. Images of the same before (upper panel) and after application of 10µl of semen sample (lower panel).
Figure 6c: BCIP (up) and NBT+BCIP (down) dyes used to test colour formation in different vaginal samples at different time points for a comparative study.
Figure 7: Schematic diagram of the present invention.
Reference Index:
1. Absorbent Pad: An absorbent pad is positioned at the end of the test strip to facilitate the migration of the sample and reagents through capillary action.
2. Test Pad: A test pad contains immobilized capture monoclonal antibodies (SEMG2) specific to semen biomarkers.
a. The presence of these biomarkers will form a coloured test line 7 on this pad.
b. A control line 6 comprising immobilized goat polyclonal antibody to rabbit IgG is present on the test pad to validate the proper functioning of the assay.
3. Conjugate Pad: A conjugate pad is coated with gold nanoparticles conjugated to detector monoclonal antibodies (SEMG2) against the semen biomarkers.
4. Sample Pad: A sample pad is present at the beginning of the test strip, where the user applies the biological sample.
5. Acid Phosphatase (ACP) Test Strip: An additional small strip coated with nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is included to detect the presence of acid phosphatase, another semen biomarker.
6. Control line
7. Test line
2A. a display window over the test pad 2
4A. a sample introducing window over the sample pad 4
5A. a sample introducing window over the ACP strip 5
Figure 8: The representation of the present invention before sample run
Figure 9: The representation of the present invention after sample run
Detail description of the Invention:
The embodiments of this invention, illustrating all its features, will now be discussed in detail.
The words "comprising," "having," "containing," and "including," and other forms thereof, are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. It must also be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Although any systems and methods similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred, systems and methods are now described. The disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms.
The present invention discloses the fabrication of a microfluidic paper-based analytical device (µPAD) following the design of lateral flow immunoassay strips. These strips will present the positive results in the form of coloured bands on specific positions.
The present invention will engage the immunological reaction between anti-semenogelin antibodies (specifically anti-SEMG2 antibodies) with the targeted antigen (Semenogelin-2) present in the semen samples. This protein is present in high amounts in male semen samples which offers gender-level specificity, excluding the chance of false detection of body fluids present in females. Hence, positive detection of SEMG2 in vaginal swabs will confirm the presence of male semen inside the female vagina which comes in times of unprotected sex or sexual assaults. In addition, the present invention will also allow simultaneous detection of semen acid phosphatase by the biochemical colourimetric reaction of converting colourless 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitrotetrazolium Blue (NBT) solution into intensified purple coloured products. The said kit will only confirm the presence of semen in an examined sample when both these tests provide positive results and form two coloured bands. Therefore, the present invention will allow multiplex detection of molecules present in the semen and thereby will improve the accuracy by reducing the occurrence of false positives, which is common in singleplex reactions.
The physical design of the present invention consists of a plastic cassette consisting of four types of fibre pads and membranes (Figure 7 and Figure 8). These pads and membranes will contain the specific antibodies and chromogenic substrates. The antigens/reactants will be supplied through the semen samples under examination. The sample under examination will be collected by cotton swabs and will be extracted in the liquid buffer, all of which will be supplied with the said kit. Sample extracted buffer will then be applied onto the sample pad 4 of the said kit. If the sample contains semen antigens/reactants, then a positive coloured bands will appear on the test pad 2 at the result displaying windows. A control line 6 will also appear to ensure a positive flow of the reactants over the applied reagents on the test pad 2. Noteworthy, the appearance of the control line 6 will be positive in both cases of the presence or absence of semen in the examined samples. Therefore, the appearance of two test lines will only confirm a positive reaction. (Figure 9)
SD Kit experimental designs and observations
The design of the present invention is based on the development of one test line and one control line 6 on the test pad 2 and a purple Acid phosphatase (AP) colour formation with semen samples on the Acid Phosphatase (ACP) Test Strip 5.
The test lines are made for detection of SEMG2 antigen in human semen sample and Acid phosphatase (AP) activity in the same. The SEMG2 test line was drawn with dispensing anti-SEMG2 antibody and the AP test line was drawn with BCIP dye on nitrocellulose membrane strips.
Sample Preparation:
Semen or vaginal secretion or vaginal secretion and semen sample is extracted in 1X Phosphate-Buffered Saline (PBS) [NaH2PO4 1mM, Na2HPO4 8.1 mM, NaCl 150 mM] before testing. Mostly 1:20 dilution of raw semen sample i.e. 50 µl of semen in 1 ml of 1X PBS is used for extraction for 1 hour at room temperature (RT). In case of the present invention the said extraction is successful within 5-15 min following the same procedure and positive results are found for freshly extracted as well as one month older extraction of the semen samples. After extraction, the samples were either readily used or stored in refrigerator at 40C. Generally, 100 µl of samples have been tested both in dipstick method and on direct dropwise application to the present invention. Swabs containing test samples were extracted directly in 500 µl PBS. The positive results are observed upto 1:100 dilutions of extracted samples to develop SEMG2 test lines on the test pad 2 and AP activity on ACP test strip 5. Positive results were found for the SEMG2 test lines only in samples containing semen (i.e. only semen extracts and vaginal secretion with semen) but was negative in test samples containing only vaginal secretion.
Nitrocellulose Membrane Selection:
A series of nitrocellulose (NC) membranes were tested for the present invention. Those were FF80 HP, FF80 HP Plus, FF120 HP, FF120 HP Plus, FF120 HP Plus Thick, FF170 HP, FF170 HP Plus, FF170 HP Plus Thick, Immunopore RP and Prima 40 from Cytiva. All these membranes were dispensed with anti SEMG2 detector antibody (abcam #ab245094; 1mg/ml) and rabbit secondary control antibody (abcam # ab6702; 2mg/ml) and thereafter was blocked with 0.5% Bovine Serum Albumin (BSA) (in 1X PBS). A strip of nitrocellulose membrane sized about 3 mm x 30 mm (width x length) was striped with antibody at a volume of 0.5 ul/3mm. However the test line antibody was dispensed at 1mg/ml and the control line 6 antibody was dispensed at 0.4 mg/ml concentration. Upon BSA treatment, strips are washed with PBS once and are dried at room temperature for 1-2 h and were stored at 4 ºC in dehumidified condition before usage. These membranes were assembled either with absorbent pad 1 only for dipstick method of sample run or with conjugate pad 3, sample pad 4 and absorbent pad 5 for drop-wise sample addition. Figure 1 showed that among all the tested types of nitrocellulose membranes, FF120 HP Plus Thick, FF170 HP, FF170 HP Plus, FF170 HP Plus Thick showed positive results in the dipstick method of semen sample (1:20) run. However, in repeated experiments, FF120 HP Plus Thick showed better performance in terms of faster sample run, for which it was selected for the next set of experiments. FF120 HP Plus Thick strips allowed the sample to reach the absorbent pads 1 (in dipstick method) in 15-20 mins while it took 40 min to be run on FF170 strips.
Gold Nanoparticle Conjugate and Conjugate Pad (CP) 4 Selection:
Two types of conjugates pads (CP) 3, ST17 and Fusion 5 from Cytiva were tested. ST17 is made of glass fibre and Fusion 5 is made of cellulose fibre. Although both pads showed positive results in dipstick sample run, ST17 glass fibre performed better in terms of quick drying and better conjugate release (Figure 2). Hence, the conjugate pad 3 selected is the ST17 glass fibre pad. It contains anti-SEMG2 capture antibody (abcam #ab244821; 1mg/ml) coated gold nanoparticles. The antibody concentration tested for gold nanoparticle conjugation ranged among 0.05, 0.1 and 0.25 mg/ml. It was conjugated with 20 nm (abcam, #ab188215), 40 nm (abcam, #ab154873) and 80 nm (abcam, #ab154876) gold conjugates by NHS linkage following manufacturer’s protocol. The conjugate pad was treated with 10% - 20% sucrose, 0.05% Tween 20 and 0.5% BSA and is dried at 37 ºC for 1-2 h before application of the conjugate (3µl for a 3 mm x 5mm pad), after which it is dried overnight at room temperature. Upon drying, the pad was either assembled in the said kit for usage or stored at 4ºC in dehumidified condition. For 20 nm, 0.025 mg/ml; for 40 nm, 0.1 mg/ml and for 80 nm, 0.05 mg/ml concentration of the capture antibody was used. The conjugates showing absorption maxima of 0.1 Absorbent Unit (AU) at 1:200 dilutions at 528 nm, 530 nm and 550 nm respectively confirmed the accuracy of gold nanoparticle conjugate development at a concentration of 20 OD. The 20 nm, 40 nm and 80 nm gold nanoparticle conjugates should form bright red, bright pink or brown lines respectively on the control and test lines of the nitro cellulose membranes for positive results on the test pad 2.
A test run with 100 ul of the 1:20 extracted semen sample following the dipstick method showed that 20 nm gold nanoparticle conjugates failed to form any bright red control or test lines and hence was discarded for later experiments. Both 40 nm and 80 nm gold nanoparticle conjugates produced positive test and control lines (Figure 3) on the test pad 2. But 40 nm was ultimately selected for its brighter (pink) intensity over the 80 nm (brown).
Freshly prepared kits were working fine at Conjugate Pad treatment buffer with 10% sucrose. However, for longer storage, 20% sucrose showed better activity as compared to 10% and 15% (produced very faint test lines). Temperature sensitivity experiments showed that the kits were more stable at 4 ºC compared to room temperature storage since they produced positive test lines at cold storage for 30 days whereas test line sensitivity declined drastically just after 7 days storage at room temperature (Figure 4). Therefore, the Conjugate Pad treatment buffer composition was finalized to be 20% sucrose, 0.05% Tween 20 and 0.5% Bovine Serum Albumin to support longer stability of the kit during storage.
Sample Pad 4 and Absorbent Pad 1 Selection:
The sample pad 4 used the ST14 glass fibre pad from Cytiva and the absorbent pad 1 used the CF7 cellulose pad from Cytiva. In each kit, a sample pad of 3 mm x 15 mm and an absorbent pad of 3 mm x 20 mm were placed at the farthest to each other maintaining the order of arrangements as follows: sample pad 4 overlapping the conjugate pad 3, overlapping the nitrocellulose membrane of the test pad 2 to be overlapped by the absorbent pad 1 at the ends (Figure 7). The length of the absorbent pad 1 is purposefully kept longer to retain more sample that would support unidirectional flow of sample from sample pad towards the absorbent pad.
Acid Phosphatase (ACP) Testing:
To test acid Phosphatase activity in semen, previously, alpha-naphthyl phosphate was used as substrate and Fast Blue BB dye was used as the chromogenic indicator. A positive dephosphorylation reaction (under acidic condition at pH 4.5) by the APase on alpha-naphthyl phosphate (in acetate buffer) can be identified by the formation of a purple product in presence of the dye. However, the dye tends to be destabilized with storage either at room temperature or in 4 ºC, forms brown precipitation and therefore fails to develop purple colouration by reacting with semen APase (Figure 5). This makes it impossible to be used as a semen indicator in stored kits. Hence, two different dye solutions, BCIP (5-Bromo-4-chloro-3-indolyl phosphate disodium salt; Sigma Aldrich, #B6149) and BCIP+NBT (5-Bromo-4-chloro-3-indolyl phosphate and Nitrotetrazolium Blue chloride, Sigma Aldrich, #B6149 and #N6876) are tested alternatively.
1X concentration of BCIP was used at 0.5 mg/ml in acetate buffer, pH 4.5) and 1X concentration of BCIP+NBT was tested at 0.165 mg/ml of BCIP; 0.33 ug/ml of NBT acetate buffer, pH 4.5). Both these solutions remain colourless until tested with semen extracts. BCIP reacts with semen APase at acidic pH (pH 4.5) to form light blue colour within 5 min and the intensity increased with higher concentration of the dye. BCIP+NBT forms purple colour at a much faster rate (3 min) when tested with semen. Similar to BCIP, the intensity of the purple colour again increased with dye concentration. These alternative dyes gave better results than Fast Blue dye, in terms of storage stability both at 4 ºC and at room temperature. They did not form brown spots even after 3 days of storage at room temperature (Figure 6a). Although, at higher concentrations (BCIP at 20X concentration and BCIP+NBT at 2.5X concentration), they lose their colourless properties, that did not interfere with the blue or purple colour formation with semen. Positive results for APase activity in 10-20 ul of 1:20 extracted semen samples as well as in vaginal secretion with semen samples were obtained both for BCIP and BCIP+NBT after 30 days of storage at 4 ºC (Figure 6b). It is noteworthy, BCIP and BCIP+NBT gave positive results also in vaginal secretion without semen (Figure 6c). However, the colour development in this case was much slower and lighter as compared to samples containing semen. Therefore, it is recommended, that acid phosphatase colour development within 5 min along with positive SEMG2 test line formation should be considered as positive indicator for the presence of semen in any tested samples.
BCIP and NBT were selected for the kit development since they formed a final colour of higher intensity and showed better storage stability than BCIP. Also, BCIP+NBT molecules were harder to be retained on nitrocellulose membranes while sample run and hence were applied on Fusion 5 and kept in separate sample loading wells (Acid Phosphatase (ACP) Test Strip 5) in the kit.
Embodiment 1
The present invention is a point-of-care diagnostic kit for detection of semen, comprising four types of fibre pads and membranes in a kit cassette:
Absorbent Pad: An absorbent pad (Material: Grade CF7) is positioned at the end of the test strip to facilitate the migration of the sample and reagents through capillary action.
Test Pad: A test pad (Material: FF120HP Plus Thick) contains immobilized capture monoclonal antibodies (SEMG2) specific to semen biomarkers. The presence of these biomarkers will form a coloured test line on this pad. A control line 6 comprising immobilized goat polyclonal antibodies to rabbit IgG is present on the test pad to validate the proper functioning of the assay.
Conjugate Pad: A conjugate pad (Material: ST17) is coated with gold nanoparticles (20 nm) conjugated to detector monoclonal antibodies (SEMG2) against the semen biomarkers.
Sample Pad: A sample pad (Material: ST14) is present at the beginning of the test strip, where the user applies the biological sample.
ACP Test Strip: An additional small strip (Material: Fusion 5) coated with NBT+BCIP (nitro-blue tetrazolium + 5-bromo-4-chloro-3-indolyl phosphate) is included to detect the presence of acid phosphatase, another semen biomarker.

Embodiment 2
The present invention is a point-of-care diagnostic kit for detection of semen, comprising four types of fibre pad and membranes in a cassette:
Absorbent Pad: An absorbent pad (Material: Grade CF7) is positioned at the end of the test strip to facilitate the migration of the sample and reagents through capillary action.
Test Pad: A test pad (Material: FF120HP Plus Thick) contains immobilized capture monoclonal antibodies (SEMG2) specific to semen biomarkers. The presence of these biomarkers will form a coloured test line on this pad. A control line 6 comprising immobilized goat polyclonal antibodies to rabbit IgG is present on the test pad to validate the proper functioning of the assay.
Conjugate Pad: A conjugate pad (Material: ST17) is coated with gold nanoparticles (40 nm) conjugated to detector monoclonal antibodies (SEMG2) against the semen biomarkers.
Sample Pad: A sample pad (Material: ST14) is present at the beginning of the test strip, where the user applies the biological sample.
ACP Test Strip: An additional small strip (Material: Fusion 5) coated with NBT+BCIP (nitro-blue tetrazolium + 5-bromo-4-chloro-3-indolyl phosphate) is included to detect the presence of acid phosphatase, another semen biomarker.

Embodiment 3
The present invention is a point-of-care diagnostic kit for detection of semen, comprising four types of fibre pads and membranes in a cassette:
Absorbent Pad: An absorbent pad (Material: Grade CF7) is positioned at the end of the test strip to facilitate the migration of the sample and reagents through capillary action.
Test Pad: A test pad (Material: FF120HP Plus Thick) contains immobilized capture monoclonal antibodies (SEMG2) specific to semen biomarkers. The presence of these biomarkers will form a coloured test line on this pad. A control line 6 comprising immobilized goat polyclonal antibodies to rabbit IgG is present on the test pad to validate the proper functioning of the assay.
Conjugate Pad: A conjugate pad (Material: ST17) is coated with gold nanoparticles (80 nm) conjugated to detector monoclonal antibodies (SEMG2) against the semen biomarkers.
Sample Pad: A sample pad (Material: ST14) is present at the beginning of the test strip, where the user applies the biological sample.
ACP Test Strip: An additional small strip (Material: Fusion 5) coated with NBT+BCIP (nitro-blue tetrazolium + 5-bromo-4-chloro-3-indolyl phosphate) is included to detect the presence of acid phosphatase, another semen biomarker.

Embodiment 4
The present invention is a point-of-care diagnostic kit for detection of semen, comprising four types of fibre pads and membranes in a cassette:
Absorbent Pad: An absorbent pad (Material: Grade CF7) is positioned at the end of the test strip to facilitate the migration of the sample and reagents through capillary action.
Test Pad: A test pad (Material: FF170HP Plus Thick) contains immobilized capture monoclonal antibodies (SEMG2) specific to semen biomarkers. The presence of these biomarkers will form a coloured test line on this pad. A control line comprising immobilized goat polyclonal antibodies to rabbit IgG is present on the test pad to validate the proper functioning of the assay.
Conjugate Pad: A conjugate pad (Material: ST17) is coated with gold nanoparticles (40 nm) conjugated to detector monoclonal antibodies (SEMG2) against the semen biomarkers.
Sample Pad: A sample pad (Material: ST14) is present at the beginning of the test strip, where the user applies the biological sample.
ACP Test Strip: An additional small strip (Material: Fusion 5) coated with NBT+BCIP (nitro-blue tetrazolium + 5-bromo-4-chloro-3-indolyl phosphate) is included to detect the presence of acid phosphatase, another semen biomarker.

As shown in figure 8 and figure 9, the said kit is housed in a plastic or waterproof cassette with a display window 2A over the test pad 2 and two sample introducing window 4A , 5A over the sample pad 4 and over the ACP strip 5 respectively.
,CLAIMS:Claims:
1. A point-of-care diagnostic kit for detection of semen, comprising four types of fibre pads and membranes based on the development of one test line 7 and one control line 6 and a purple Acid phosphatase (AP) color formation with semen samples:
a. Absorbent Pad 1,
b. Test Pad 2,
c. Conjugate Pad 3,
d. Sample Pad 4, and
e. Acid Phosphatase (ACP) Test Strip 5;
wherein
said Absorbent Pad 1 is positioned at the end of the test strip to facilitate the migration of the sample and reagents through capillary action,
said Test Pad 2 contains immobilized capture monoclonal antibodies (SEMG2) specific to semen biomarkers to form a coloured test line on this pad,
said Conjugate Pad 3 is coated with (20 nm – 80nm) gold nanoparticles conjugates to detector monoclonal antibodies (SEMG2) against the semen biomarkers,
said Sample Pad 4 is present at the beginning of the test strip, where the user applies the biological sample,
said Acid Phosphatase Test Strip 5, an additional small strip is coated with nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to detect the presence of acid phosphatase, another semen biomarker.
2. The diagnostic kit as claimed in claim 1, wherein the said absorbent pad 1 is made of Grade CF7 cellulose pad.
3. The diagnostic kit as claimed in claim 1, wherein the said test pad 2 is made of nitrocellulose membranes, preferably FF120 HP Plus Thick, FF170 HP, FF170 HP Plus, FF170 HP Plus Thick.
4. The diagnostic kit as claimed in claim 1, wherein the said conjugate pad 3 is selected from ST17 glass fibre and Fusion 5 cellulose fibre.
5. The diagnostic kit as claimed in claim 4, wherein the said conjugate pad 3 contains (0.05- 0.25) mg/ml anti-SEMG2 capture antibody coated gold nanoparticles ranging from (20nm- 80nm).
6. The diagnostic kit as claimed in claim 4, wherein the said conjugate pad 3 is treated with buffer composition comprising 10% - 20% sucrose, 0.05% Tween 20 and 0.5% Bovine Serum Albumin, dried at 37 ºC for 1-2 h before application of the gold nanoparticle conjugate (3µl for a 3 mm x 5mm conjugate pad 3) and then it is dried overnight at room temperature.
7. The diagnostic kit as claimed in claim 1, wherein a control line comprising immobilized goat polyclonal antibodies to rabbit IgG is created on the test pad 2 to validate the proper functioning of the assay.
8. The diagnostic kit as claimed in claim 1, wherein the said sample pad 4 is be made of ST14 glass fibre.
9. The diagnostic kit as claimed in claim 1, wherein the said ACP Test Strip 5 may be made of Fusion 5 cellulose pad coated nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.
10. The diagnostic kit as claimed in claim 1, wherein the said kit is housed in a plastic or waterproof cassette with a display window 2A over the test pad 2 and two sample introducing window 4A , 5A over the sample pad 4 and over the ACP strip 5 respectively.
11. The diagnostic kit as claimed in claim 1, wherein the said sample pad 4 of 3 mm x 15 mm and the said absorbent pad 1 of 3 mm x 20 mm were placed at the farthest to each other maintaining the order of arrangements as follows: sample pad 4 overlapping the conjugate pad 3, overlapping the nitro cellulose membrane to be overlapped by the absorbent pad 1 at the ends
12. The diagnostic kit as claimed in claim 1, wherein the time taken by the sample to reach the absorbent pads is 15-20 mins in a single lateral flow assay.