SPERM ANTIBODY Elisa                     

BL-48-E

FOR IN VITRO DIAGNOSTIC USE

1       Introduction

Antibodies directed against spermatozoa antigens may cause infertility in women or men. The application of the Anti-Spermatozoa Antibody ELISA from Bio-Line 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 20% of these cases the presence of anti-spermatozoa antibodies in the male or the female patient is detectable (1).

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 exert heterogeneous effects on the ability of spermatozoa to fertilize. The inhibiting effect of anti-spermatozoa antibodies on the motility of spermatozoa by binding to their surface and by agglutinating processes is well-known (2).

The penetration of the spermatozoa into the cervical mucus is impaired by the presence of anti-spermatozoa antibodies in the seminal plasma and/or in the cervical mucus (3). Anti-spermatozoa antibodies negatively influence the capacitation and the acrosome reaction of spermatozoa and thereby impede the interaction of the spermatozoa with the oocyte (4).

The interaction of the spermatozoon with the oocyte and the subsequent binding to and penetration of the zona pellucida may be inhibited by anti-spermatozoa antibodies. The following fusion of the oocyte and a spermatozoon may also be impaired by the presence of anti-spermatozoa antibodies (5).

According to Crosignani et al. (6), the rate of pregnancies in couples with anti-spermatozoa antibodies on the part of the man or the woman are 38% lower compared to the control groups. Furthermore an influence on the implantation and on the early embryological development could be confirmed. An association of anti-spermatozoa antibodies and miscarriages is discussed.

The frequency of anti-spermatozoa antibodies in infertile couples amounts to 20% (7).

Anti-spermatozoa antibodies may occur dissolved in the ejaculate or bound to the surface of spermatozoa. Anti-spermatozoa antibodies may be found in men and in women (8). In women anti-spermatozoa antibodies 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 (9).

2       PRINCIPLE of the test

The anti-spermatozoa antibody ELISA (Enzyme Linked ImmunoSorbent Assay) from Bio-Line is a solid-phase sandwich enzyme-immunoassay for the quantitative determination of anti-spermatozoa antibodies in human serum.

The ELISA-plate is coated with a mix of spermatozoa proteins which are recognized by anti-spermatozoa antibodies. The samples and calibrators are pipetted into the wells and then incubated. During this incubation anti-spermatozoa antibodies bind to the spermatozoa proteins and are thus immobilised on the plate. After washing the enzyme conjugate, consisting of anti-human globulin antibodies covalently coupled to horseradish peroxidase, is added. After removal of the unbound conjugate by washing the horseradish peroxidase oxidizes the then added substrate TMB (3,3’,5,5’-tetramethylbenzidine) yielding a colour reaction which is stopped with 0.25 M sulphuric acid (H₂SO₄). The extinction is measured at a wavelength of 450 nm with a microplate reader. The use of a reference measurement with a wavelength >550 nm is recommended.

3       Precautions

·       This kit is for in vitro diagnostic use only.

·       For information on hazardous substances included in the kit please refer to Material Safety Data Sheets.

·       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.

·       Avoid contact with Stop Solution containing 0.25 M H2SO4. It may cause skin irritation and burns.

·       Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.

·       Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.

·       Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may give false results.

·       Handling should be in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.

·       Do not use reagents beyond expiry date as shown on the kit labels.

·       All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes.

·       Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of different plates even if 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.

·       Chemicals and prepared or used reagents have to be treated as hazardous waste according the national biohazard safety guideline or regulation.

·       Safety Data Sheets for this product are available upon request directly from Bio-Line.
The Safety Data Sheets fit the demands of: EU-Guideline 91/155 EC.

4       Kit Components

4.1  Contents of the Kit

1.   microplate                         12x8 (break apart) strips
96 wells
Wells coated with Sperm antigen

2.   standard curve                N=1 to 4
4 vials, 0.5 ml
ready to use
Calibrator 1 – colourless screw cap
Calibrator 2– white screw cap
Calibrator 3 – yellow screw cap
Calibrator 4 – blue screw cap

3.   standard zero                  1 vial, 50 ml
ready to use
0 U/ml

4.   Enzyme Conjugate           1 vial, 5 ml
ready to use

5.   chromogene                     1 vial, 13 ml
Ready to use
TMB

6.   stop solution                     1 vial, 12 ml
Ready to use
Contains 0.25M H₂SO₄
Avoid contact with the stop solution. It may cause skin irritations and burns.

7.   wash solution                  1 vial, 50 ml (10X concentrated)
see “Preparation of Reagents“

8.   control                              N=1, 1 vial, 0.5 ml (green screw cap)
equivalent to 100-200 U/ml

Note: Additional Zero Calibrator for Sample dilution available on request.  

4.2  Equipment and material required but not provided

1.     A microtiterplate calibrated reader (450±10 nm).

2.     Calibrated variable precision micropipettes (Varipette Eppendorf), Multipette Eppendorf or similar products.

3.     Absorbent paper.

4.     Distilled water.

4.3  Storage and stability of the Kit

·       When stored at 2° to 8°C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.

·       Enzyme-Conjugate, Substrate Solution, Calibrators and Zero Calibrator must be stored at 2° to 8°C.

·       Microtiter wells must be stored at 2° to 8°C. Once the foilbag has been open care should be taken to close it tightly again.

4.4  Preparation of Reagents

Allow all reagents and required number of strips to reach room temperature prior to use.

Wash Solution

Dilute the concentrated washing solution (50 ml) by adding 450 ml distilled or deionised water. The diluted washing solution is stable for 4 weeks at refrigerator temperatures (4 °C – 8 °C / 39 °F – 46 °F). Attention: Do not use unpurified tap water!

4.5  Disposal of the Kit

The disposal of the kit must be made according to the national official regulations. Special information for this product are given in the Material Safety Data Sheets.

4.6  Damaged Test Kits

In case of any severe damage of the test kit or components, Bio-Line have to be informed written, latest one week after receiving the kit. Severely 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 according to the official regulations.

5       SPECIMEN

5.1  Specimen collection

Sample Material: Serum

Collect blood by venipuncture, allow to clot, and separate serum by centrifugation at room temperature.

Do not use haemolytic, icteric or lipaemic serum.

5.2  Specimen storage

Samples may be stored at different temperatures for the following time-spans:

–         Environmental temperature up to 30 °C (86 °F):                                      up to three days

–         Refrigerator temperature (2 – 8 °C / 36 °F – 46 °F):                               up to one week

–         Household freezer temperature (-10 °C – -20 °C / 14 °F – -4 °F):          up to one year

In case you want to effect the test with seminal plasma please inquire for the actual procedures.

5.3  Specimen dilution

Dilute sera 1: 100 with dilution buffer (1:100 dilution: 5 µl of serum + 495 µl of dilution buffer).

6       test procedure

6.1  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 pipet tips for each calibrator, control of sample in order to avoid crosscontamination.

·       Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents be ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption.

6.2   Procedural Notes

·       All calibrators, samples, and controls should be run in duplicate concurrently so that all conditions of testing are the same.

·       At temperatures higher than 30 °C (86 °F) the samples should be transported cooled or refrigerated.

·       The time to stop the (enzymatic colour) reaction may have to be adjusted (shortened).

·       Severely haemolytic or lipaemic sera or sera from patients with liver diseases should not be used. Results may be adversely affected by certain pathologic conditions, such as poly- and monoclonal gammapathies, autoimmune diseases or by an altered immune status.

6.3   Assay Procedure

1.     Secure the desired number of Microtiterwells in the holder.

2.     Dilute sera 1: 100 with dilution buffer (1:100 dilution: 5 µl of serum + 495 µl of dilution buffer).

3.     Dispense 50 µl of Sperm Antibody Calibrators with new disposable tips into appropriate wells.

4.     Dispense 50 µl of diluted serum with new disposable tips into the respective wells. Time between distribution of first Calibrator and last sample can be up to 10 minutes without affecting the results.

5.     Incubate for 1 hour at 37°C.

6.     Briskly shake out the contents of the wells.
Rinse the wells 3 times with diluted Wash Solution (200 µl per well). Strike the wells sharply on absorbent paper to remove residual water droplets.
Important note:
The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure!

7.     Dispense 50 µl Enzyme Conjugate into each well.

8.     Incubate for 1 hour at 37°C.

9.     Briskly shake out the contents of the wells.
Rinse the wells 5 times with diluted Wash Solution (200 µl per well). Strike the wells sharply on absorbent paper to remove residual water droplets

10.  Add 50 µl of Substrate Solution to each well immediately after the washing of the wells.

11.  Incubate for 30 minutes at room temperature.

12.  Stop the enzymatic reaction by adding 50 µl of Stop Solution to each well.

13.  Read the OD at 450±10 nm with a microtiterplate reader within 10 minutes after adding the Stop Solution.

Since calibrators are assayed in each run, absorbance fluctuations do not affect the absolute results. In any case it is highly recommended to use an additional internal control if available.

Pipetting Scheme for the Sperm Antibody ELISA

In this pipetting scheme the recommended positions for the blank (please use the dilution buffer included in this kit), calibrators (S1 – S4), positive control (PC) and for the patient samples (P1 – P42) are shown as double determinations.

6.4  Calculation of Results

1.     Calculate the average absorbance values for each set of calibrators, controls and patient samples.

2.     Construct a calibrator curve by plotting the mean absorbance obtained from each calibrator against its concentration with absorbance value on the vertical(Y) axis and concentration on the horizontal (X) axis.

3.     Using the mean absorbance value for each sample determine the corresponding concentration of Estriol in ng/ml from the calibrator curve. Depending on experience and/or the availability of computer capability, other methods of data reduction may be employed.

4.     Automated method: Computer programs using cubic spline, 4 PL (4 Parameter Logistics) or Logit-Log can generally give a good fit.

7       Assay Characteristics

7.1  Expected values

Normal values:                           0 – 60 U/ml

Elevated values:                         above 60 U/ml

In the case of a value in the range near the cut-off (55 to 65 U/ml) we recommend a follow-up determination using a new sample taken within the next two weeks.

7.2  Specificity and Sensitivity

7.4  Accuracy

Quality Control

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 stated on the QC sheet always refer to the current kit lot and should be used for direct comparison of the results.

It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy 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 Bio-Line directly.

7.5  Precision

Intra and Inter Assay Variation

The within assay variability is shown below:

7.5.1  Intraassay variation coefficient:            6.88% (5.90 – 7.81 %)

For the determination of the intraassay variation coefficient 6 kits from 6 different batches (produced on different days) were used. One patient sample (optical density about 1.0) was applied 96 times per testing procedure.

7.5.2  Interassay variation coefficient                              6.45% (4.84 – 7.52 %)

For the determination of the interassay variation coefficient one strip each of 12 kits stemming from 6 different batches (produced on different days) were used. One patient sample (optical density about 1.0) was applied 72 times per testing procedure.

8       Limitations of Use

8.1  Interfering Substances

Any improper handling of samples or modification of this test might influence the results. Interferences caused by improper sample handling are explained in the chapters ‘Specimen - Collection’.

8.2  High-Dose-Hook Effect

No hook effect was observed in this test.

9       Legal Aspects

9.1  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.

9.2  Therapeutical Consequences

Therapeutical consequences should never be based on laboratory results alone even if all test results are in agreement with the items as stated under point 9.1. 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 therapeutical consequences be derived.

The test result itself should never be the sole determinant for deriving any therapeutical consequences.

9.3  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 9.2. 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.

10    REFERENCES

1.     Lahteenmaki A et al: Hum Reprod (1995) 10, 2824-28; Nagy ZP et al: Hum Reprod (1995) 10, 1775-80.

2.     Zouari R et al: Fertil Steril (1993) 59, 606-12.

3.     Eggert-Kruse W et al: Hum Reprod (1993) 8, 1025-31.

4.     Francavilla F et al: Front Biosci (1999): 1;4:9-25; Bohring C et al.: Hum Reprod (2001) 7:113-8.

5.     Mazumdar S et al.: Fertil Steril (1998) 70, 799-810; Kutteh WH: Hum Reprod, (1999) 14, 2426-9.

6.     (Crosignani et al.: PG et al.: Hum Reprod (1998) 13, 2025-32.

7.     (Lahteenmaki A et al.: Hum Reprod (1995) 10, 2824-28; Nagy ZP et al.: Hum Reprod (1995) 10, 1775-80.

8.     (Clarke GN et al.: Am J Reprod Immunol Microbiol (1985) 7, 143-7.

9.     (Abshagen K et al.: Fertil Steril (1998) 70, 355-6.

11                 Sperm Antibody Flow chart