EIA-4249 DRG Sperm Antibody Instruction Manual

EIA-4249 DRG Sperm Antibody

Product Specifications

• Product Name: Sperm Antibody (seminal plasma) ELISA EIA-4249
• Manufacturer: DRG International, Inc., USA
• Intended Use: Enzyme immunoassay for the quantitative measurement of antibodies against human spermatozoa in seminal plasma

FAQ

• Q: How should I store the kit when not in use?
  • A: Store the kit as per the manufacturer’s instructions provided in the user manual to maintain reagent stability.
• Q: Can this kit be used for testing samples other than seminal plasma?
  • A: No, this kit is specifically designed for testing antibodies against human spermatozoa in seminal plasma and should not be used for other sample types.

Please use only the valid version of the Instructions for Use provided with the kit.

INTENDED USE

The DRG Sperm Antibody (seminal plasma) ELISA is an enzyme immunoassay for the quantitative in vitro diagnostic measurement of antibodies directed against human spermatozoa in seminal plasma.

Summary and Explanation

• Antibodies directed against spermatozoa antigens may cause infertility in women or men. The application of the Sperm Antibody ELISA is recommended for the diagnosis of immunologically caused disorders of fertility.
• Unwanted childlessness is a growing problem with which up to 20% of all couples in the reproductive age are confronted temporarily or long-term. In 5-20 % of these cases, the presence of anti-spermatozoa antibodies in the male or the female patient is detectable [1,2,15].
• The definition of infertility according to the WHO (WHO Laboratory Manual for the Examination of Human Semen and Semen Cervical-Mucus Interaction, 1999) is the absence of a conception within 12 months of unprotected intercourse. The main cause of an immunological fertility disorder is the formation of antibodies directed against spermatozoa antigens.
• Anti-spermatozoa antibodies (ASA) exert heterogeneous effects on the ability of spermatozoa to fertilize. The inhibiting effect of ASA on the motility of spermatozoa by binding to their surface and by agglutinating processes is well-known [3].
• The penetration of the spermatozoa into the cervical mucus is impaired by the presence of ASA in the seminal plasma and/or in the cervical mucus [4]. ASA negatively influence the capacitation and the acrosome reaction of spermatozoa, and thereby impede the interaction of the spermatozoa with the oocyte [5,6].
• The interaction of the spermatozoon with the oocyte and the subsequent binding to and penetration of the zona pellucida may be inhibited by ASA. The following fusion of the oocyte and a spermatozoon may also be impaired by the presence of ASA [7,8].
• The rate of pregnancies in couples with ASA on the part of the man or the woman was shown to be 38% lower compared to the control groups [9]. Furthermore, an influence on the implantation and on the early embryological development could be confirmed.
• An association of ASA and miscarriages is discussed.
• The frequency of ASA in infertile couples amounts to 20% [10,11].
• ASA may occur dissolved in the ejaculate or bound to the surface of spermatozoa. ASA may be found in men and in women [12]. In women, ASA may be found in cervical mucus, oviduct liquid and follicular liquid. Men having more than 50% of their spermatozoa coated with anti-spermatozoa antibodies show a conspicuously reduced rate of fertility [13].
• ASA have been shown to be associated with chronic prostatitis which has a negative effect on male reproductive function [14].

PRINCIPLE OF THE TEST

• The DRG Sperm Antibody ELISA is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle.
• The microtiter wells are coated with a mix of spermatozoa proteins.
• During incubation, anti-spermatozoa antibodies in the samples (standards, quality control, patient specimen) bind to the coated surface of the wells.
• A washing step removes unbound sample components.
• Added enzyme conjugate binds to the immobilized antigen-antibody-complexes.
• The conjugate contains anti-human immunoglobulin antibodies, labelled with horseradish peroxidase (HRP).
• After a washing step to remove all unbound substances, the solid phase is incubated with the substrate solution. The colorimetric reaction is stopped by addition of stop solution, and optical density (OD) of the resulting yellow product is measured. The intensity of color is proportional to the concentration of the analyte in the sample.
• A standard curve is constructed by plotting OD values against concentrations of standards, and concentrations of unknown samples are determined using this standard curve.

PRECAUTIONS

WARNINGS AND PRECAUTIONS

1. This kit is for in vitro diagnostic use only. For professional use only.
2. All reagents of this test kit which contain human serum or plasma have been tested and confirmed negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should be treated as potential biohazards in use and for disposal.
3. Before starting the assay, read the instructions completely and carefully. Use the valid version of instructions for use provided with the kit. Be sure that everything is understood.
4. The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil pouch and used in the frame provided.
5. Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step.
6. Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir for dispensing a substrate solution that had previously been used for the conjugate solution may turn solution coloured. Do not pour reagents back into vials as reagent contamination may occur.
7. Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
8. Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
9. Allow the reagents to reach room temperature (20 °C to 26 °C) before starting the test. Temperature will affect the optical density readings of the assay. However, values for the patient samples will not be affected.
10. Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
11. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
12. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may give false results.
13. Handling should be done in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.
14. Do not use reagents beyond expiry date as shown on the kit labels.
15. All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes and microtiter plate readers.
16. Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of different plates even of the same lot. The kits may have been shipped or stored under different conditions and the binding characteristics of the plates may result slightly different.
17. Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
18. Some reagents contain Proclin 300, BND and/or MIT as preservatives. In case of contact with eyes or skin, flush immediately with water.
19. TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant volume of water and skin with soap and abundant water. Wash contaminated objects before reusing them. If inhaled, take the person to open air.
20. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard safety guideline or regulation.
21. For information on hazardous substances included in the kit please refer to Safety Data Sheets. Safety Data Sheets for this product are available upon request directly from DRG.

REAGENTS

Materials provided

plate

Coated with a mix of spermatozoa proteins

| Ready to use

Standard (Standard 1 – 4)

| ****

4 vials x 0.5 mL

| **Standards ***

Concentrations: 31 ‒ 62 ‒ 125 ‒ 250 U/mL

| ****

Ready to use

Quality Control

| ****

1 x 0.5 mL

| Controls *

For control values and ranges please refer to vial label or Certificate of Analysis (CoA)

Contains material of animal origin;

| ****

Ready to use

Dilution Buffer     / Zero Standard| 1 x 50 mL| **Sample Diluent ***| Ready to use

Enzyme Conjugate

| ****

1 x 8 mL

| **Enzyme Conjugate ***

Anti-human poly Ig conjugated to horseradish peroxidase Colored red.

Contains material of animal origin;

| ****

Ready to use

Substrate Solution

| ****

1 x 14 mL

| Substrate Solution

Contains 3,3‘,5,5‘-tetramethylbenzidine (TMB).

Keep away from direct sun light.

| ****

Ready to use

Stop Solution

| ****

1 x 14 mL

| Stop Solution

Contains < 5 % H2SO4. Avoid contact with the stop solution. It may cause skin irritations and burns.

| ****

Ready to use

Wash Solution| 1 x 30 mL| ****

u

Wash Solution, 40X concentrate

| See “Reagent Preparation“.
 | 1 x| Cover foil|
 | 1 x| Instructions for Use|
 | 1 x| Certificate of Analysis (CoA)|

• Contain(s) < 0.0015 % CMIT/ MIT (3:1)

u Contain(s) 0.0108 % CMIT/ MIT (3:1)

Abbreviations:

CMIT:      5-chloro-2-methyl-4-isothiazolin-3-one MIT: 2-methylisothiazol-3(2H)-one

BND:       5-Bromo-5-nitro-1,3-dioxane

Materials required but not provided

• A calibrated microtiter plate reader (450 nm, with reference wavelength at 620 nm to 630 nm) (e.g. the DRG Instruments Microtiter Plate Reader)
• Incubator for 37 °C
• Calibrated variable precision micropipettes
• Absorbent paper
• Distilled water
• Timer
• Graph paper or software for data reduction

Storage Conditions

• When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.
• Opened reagents must be stored at 2 °C to 8 °C. Microtiter wells must be stored at 2 °C to 8 °C. Once the foil bag has been opened, care should be taken to close it tightly again. Opened kits retain activity for 4 weeks if stored as described above.

Reagent Preparation

Wash Solution

• Add distilled water to the 40X concentrated Wash Solution.
• Dilute 30 mL of concentrated Wash Solution with 1170 mL distilled water to a final volume of 1200 mL.

Disposal of the Kit

• The disposal of the kit and all used materials/reagents must be performed according to the national regulations. Special information for this product is given in the Safety Data Sheet, section 13.

Damaged Test Kits

In case of any damage to the test kit or components, DRG must be informed in writing, at the latest one week after receiving the kit. Damaged single components should not be used for a test run. They have to be stored until a final solution has been found. After this, they should be disposed of according to the official regulations.

SPECIMEN COLLECTION AND PREPARATION

Seminal plasma can be used in this assay.

Note: Samples containing sodium azide should not be used in the assay.

Specimen Collection

• Collect fresh ejaculate, centrifuge at room temperature and take the supernatant (seminal plasma).

Specimen Storage and Preparation

• Seminal plasma should be capped and may be stored for up to 7 days at 2 °C to 8 °C prior to assaying.
• Specimens stored for a longer time (up to 12 months) should be frozen only once at -20 °C prior to assay. Thawed samples should be inverted several times prior to testing.

Specimen Dilution

• Prior to assaying, dilute each sample (seminal plasma) 1:5 with Dilution Buffer.

Example:

• Dilution 1:5: 100 μL sample + 400 μL Dilution Buffer (mix thoroughly)

Note: The Quality Control is ready to use and must not be diluted!

ASSAY PROCEDURE

General Remarks

• All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming.
• Once the test has been started, all steps should be completed without interruption.
• Use new disposal plastic pipette tips for each standard, quality control or sample in order to avoid cross contamination.
• Optical density is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption.
• As a general rule the enzymatic reaction is linearly proportional to time and temperature.

Test Procedure

Each run must include a standard curve. Bring all reagents and required number of strips to room temperature (20 °C to 26 °C) prior to use.

1. Secure the desired number of Microtiter wells in the frame holder.
2. Dispense 50 μL of each Zero Standard, Standard, Quality Control and diluted sample with new disposable tips into appropriate wells.
3. Cover with foil and incubate for 60 minutes at 37 °C.
4. Rinse the wells 3 times with 400 μL diluted Wash Solution per well, if a plate washer is used. – OR – Briskly shake out the contents of the wells. Rinse the wells 3 times with 300 μL diluted Wash Solution per well for manual washing. Strike the wells sharply on absorbent paper to remove residual droplets. Important note: The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure!
5. Dispense 50 μL Enzyme Conjugate into each well.
6. Cover with foil and incubate for 60 minutes at 37 °C.
7. Rinse the wells 5 times with 400 μL diluted Wash Solution per well, if a plate washer is used. – OR – Briskly shake out the contents of the wells. Rinse the wells 5 times with 300 μL diluted Wash Solution per well for manual washing. Strike the wells sharply on absorbent paper to remove residual droplets.
8. Add 50 μL of Substrate Solution to each well.
9. Incubate for 30 minutes at room temperature.
10. Stop the enzymatic reaction by adding 100 μL of Stop Solution to each well.
11. Measure the optical density (OD) of the solution in each well at 450 nm (measurement wavelength) and at 620 nm or 630 nm (reference wavelength for recommended background subtraction) with a microtiter plate reader. It is recommended that the wells be read within 10 minutes after adding the Stop Solution.

Calculation of Results

1. Calculate the average optical density (OD) values for each set of standards, quality controls and patient samples.
2. Using graph paper, construct a standard curve by plotting the mean OD obtained from each standard against its concentration with OD value on the vertical (Y) axis and concentration on the horizontal (X) axis.
3. Using the mean OD value for each sample determine the corresponding concentration from the standard curve.
4. Automated method: The results in the Instructions for Use have been calculated automatically using a 4-Parameter curve fit (4-Parameter Rodbard or 4-Parameter Marquardt are the preferred methods.). Other data reduction functions may give slightly different results.
5. The concentration of the samples can be read directly from this standard curve. The standards are already pre-diluted, therefore the 1:5 dilution of the samples must not be taken into account for the final calculation of sample concentrations.

Example of Typical Standard Curve

The following data is for demonstration only and cannot be used in place of data generations at the time of assay.

EXPECTED NORMAL VALUES

It is strongly recommended that each laboratory should determine its own normal and abnormal values.

• Normal values 0 U/mL ‒ 55 U/mL
• Borderline 55 U/mL ‒ 65 U/mL
• Elevated values > 65 U/mL

In case of values in the range near the cut-off (55 U/mL to 65 U/mL), we recommend a follow-up determination using a new sample taken within the next two weeks.
The results alone should not be the only reason for any therapeutic consequences. The results should be correlated to other clinical observations and diagnostic tests.

QUALITY CONTROL

• Good laboratory practice requires that controls be run with each calibration curve. A statistically significant number of controls should be assayed to establish mean values and acceptable ranges to assure proper performance.
• It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day to day validity of results. Use controls at both normal and pathological levels.
• The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to the kit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for direct comparison of the results.
• Employ appropriate statistical methods for analysing control values and trends. If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid.
• In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods.
• After checking the above mentioned items without finding any error contact your distributor or DRG directly.

Performance Characteristics

Detection Capability

Calculated according to CLSI guideline EP17-A2:2012.

Measuring range| 9.989 ‒ 250 U/mL

Repeatability

• For the determination of the repeatability, 4 samples were measured in 12 replicates per run.
  • Mean CV: 2.8 % (range from 2.4 % – 3.8 %)

Reproducibility (between run)

• For the determination of the inter-assay precision one strip each of 12 kits stemming from 6 different batches (produced on different days) were used. One patient sample (OD > 1.0) was applied 72 times per testing procedure.
  • Mean CV: 7.15 % (range from 6.04 % – 8.21 %)

LIMITATIONS OF USE

• Reliable and reproducible results will be obtained when the assay procedure is performed with a complete understanding of the package insert instruction and with adherence to good laboratory practice.
• Any improper handling of samples or modification of this test might influence the results.

LEGAL ASPECTS

Reliability of Results

• The test must be performed exactly as per the manufacturer’s instructions for use. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or laws.
• This is especially relevant for the use of control reagents. It is important to always include, within the test procedure, a sufficient number of controls for validating the accuracy and precision of the test.
• The test results are valid only if all controls are within the specified ranges and if all other test parameters are also within the given assay specifications. In case of any doubt or concern please contact DRG.

Therapeutic Consequences

• Therapeutic consequences should never be based on laboratory results alone even if all test results are in agreement with the items as stated under point 11.1. Any laboratory result is only a part of the total clinical picture of a patient.
• Only in cases where the laboratory results are in acceptable agreement with the overall clinical picture of the patient should therapeutic consequences be derived.
• The test result itself should never be the sole determinant for deriving any therapeutic consequences.

Liability

• Any modification of the test kit and/or exchange or mixture of any components of different lots from one test kit to another could negatively affect the intended results and validity of the overall test.
• Such modification and/or exchanges invalidate any claim for replacement.
• Claims submitted due to customer misinterpretation of laboratory results subject to point 11.2 are also invalid. Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the test kit.
• Any damage caused to the test kit during transportation is not subject to the liability of the manufacturer.

REFERENCES

1. Lahteenmaki A et al. Hum Reprod (1995) 10, 2824-28
2. Nagy ZP et al. Hum Reprod (1995) 10, 1775-80.
3. Zouari R et al. Fertil Steril (1993) 59, 606-12
4. Eggert-Kruse W et al. Hum Reprod (1993) 8, 1025-31
5. Francavilla F et al. Front Biosci (1999) 4, 9-25
6. Bohring C et al. Hum Reprod (2001) 7,113-8
7. Mazumdar S et al. Fertil Steril (1998) 70, 799-810
8. Kutteh WH. Hum Reprod (1999) 14, 2426-9
9. Vegetti Wet al. Hum Reprod (1998) 13, 1796-800
10. Lahteenmaki A et al. Hum Reprod (1995) 10, 2824-28
11. Nagy ZP et al. Hum Reprod (1995) 10, 1775-80
12. Clarke GN et al. Am J Reprod Immunol Microbiol (1985) 7, 143-7
13. Abshagen K et al. Fertil Steril (1998) 70, 355-6
14. Jiang Y et al. J Reprod Immunol (2016) 118:85-91
15. Lu SM et al. Asian J Androl (2019) 21(5):473-477
16. Clarke GN. Fertility and Sterility. (2009) 91(2) :639-643
17. Barbonetti A et al. Human Reproduction (2019) 34(5) : 834–841
18. Gatimel N et al. RBMO (2018) 00(0): 1-7.
19. Silva CA et al. Clinic Rev Allerg Immunol (2012) 42:256-263

SYMBOLS USED

| European Conformity
---|---
| Consult instructions for use *
***| In vitro diagnostic medical device
| Catalog number *
| Batch code *
***| Contains sufficient for tests
| Temperature limit
| Use-by date
| Manufacturer
| Distributor
| Date of manufacture
| Biological risks
*| Caution
***| Unique device Identifier
 |
| For research use only
Distributed by| Distributed by
Content| Content
Volume/No.| Volume / No.
 |
Microtiterwells**| Microtiterwells
Antiserum| Antiserum
Enzyme Conjugate| Enzyme Conjugate
Enzyme Complex| Enzyme Complex
Substrate Solution| Substrate Solution
Stop Solution| Stop Solution
Zero Standard| Zero Standard
Standard| Standard
Control| Control
Assay Buffer| Assay Buffer
Wash Solution| Wash Solution
1N NaOH| 1N NaOH
1 N HCl| 1 N HCl
Sample Diluent| Sample Diluent
Conjugate Diluent| Conjugate Diluent

Contacts

DRG Instruments GmbH, Germany

• Frauenbergstraße 18, 35039 Marburg
• Phone: +49 (0)6421-1700 0,
• Fax: +49 (0)6421-1700 50
• Website: www.drg-diagnostics.de
• E-mail: drg@drg-diagnostics.de

DRG International, Inc., USA

• 841 Mountain Ave., Springfield, NJ 07081
• Phone: 973-564-7555, Fax: 973-564-7556
• Website: www.drg-international.com
• E-mail: corp@drg-international.com

References

• Analyticon® Biotechnologies GmbH

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