PCS

A. Overview
Human Surfactant Protein D (SP-D) is a member of the collageneous subfamily of glycoproteins and
calcium-dependent lectins (collectins). SP-D is a homotrimeric protein consisting of three 43kDa units, that are bonded at their N-termini. Most preparations of SP-D contain predominantly dodecamers (four trimeric subunits), but also higher multimers have been observed. Each unit consists of at least four discrete structural domains: a short N-terminal domain; a relatively long collagenous domain, a short amphipathic connecting peptide, and a C-terminal, Ctype lectin carbohydrate recognition domain (CRD).
SP-D is synthesized and secreted by two types of non-ciliated epithelial cells in the peripheral airway,
alveolar type II cells and Clara cells. It is also expressed by various epithelial cells in the gastrointestinal and genitourinary tracts. In the lungs, SP-D participates in the innate response to inhaled microorganisms and organic antigens. SP-D binds to surface glycoconjugates of various microorganisms and to
oligosaccharides associated with the surface of numerous organic antigens.
Studies have shown that SP-D expression may be related to a number of human diseases: cystic fibrosis,
acute interstitial pneumonias (ARDS), asthma, bronchopulmonary dysplasia, alveolar proteinosis etc.
Animal studies have shown increased levels of SP-D expession in cases of silicosis and hyperoxia (in rat
models) as well as IL-4 overexpression and invasion of infectious agents (in mice models)..

B. Test Principle
In the Human Surfactant Protein D ELISA, calibrators, quality controls and samples are incubated in
microtitration wells coated with a monoclonal anti-human Surfactant Protein D antibody. After overnight
incubation and a washing, the HRP conjugate is added and incubated two hours with captured Surfactant
Protein D. After a thorough wash, the remaining conjugate is allowed to react with the H2O2 tetramethylbenzidine substrate solution. The reaction is stopped by addition of sulfuric acid solution and absorbance of the resulting yellow colour product is measured spectrophotometrically at 450 nm. The absorbance is proportional to the Surfactant Protein D concentration. A standard curve is constructed by
plotting absorbance values versus concentrations of Surfactant Protein D standards, and the concentrations in unknown samples are determined using this standard curve.

C. Additional Required Materials
Test tubes for diluting samples
Precision pipettes to deliver 10-1000 µl and disposable tips
Multichannel pipette 50-200 µl and disposable tips
Microplate reader with 450 ± 10 nm filter
Software package facilitating data generation and analysis
Orbital microplate shaker capable of approximately 300 rpm (optional)
Microtitration plate washer (optional) [Manual washing is possible but not preferable.]
Absorbent material for blotting the microtiter plate
Glassware (graduated cylinder and bottle for Wash Solution)
Deionized (distilled) water

D. Procedural Guidelines
For in vitro research use only.
Avoid contact with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide. Wear gloves and eye protection when handling these reagents. In case of contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medical attention, if necessary.
Wear gloves and laboratory coats when handling immunodiagnostic materials.
The materials must not be pipetted by mouth.
Do not drink, eat or smoke in the areas where immunodiagnostic materials are being
handled.
Reagents with different lot numbers should not be mixed.
Reagents should not be used beyond the expiration marked on kit label.

E. List of component
Component                                                                                              Amount
Microtiter Strips coated with anti-Surfactant Protein D antibody                96 wells
Conjugate Solution (Anti- Surfactant Protein D Antibody,
F(ab‘)2 x HRP Conjugate), concentrated                                                   0.15 ml
Recombinant SP-D Standards :1.56, 3.13, 6.25, 12.5, 25, 50, and 100
ng/ml                                                                                                          7x2 vials
Quality Control High                                                                                   2 vials
Quality Control Low                                                                                   2 vials
Dilution Buffer Concentrate (4x)                                                                  2x12.5 ml
Wash Solution Concentrate (10x)                                                               50 ml
Substrate Solution (TMB)                                                                           13 ml
Stop Solution                                                                                              13 ml

F. Preparation of reagents and samples
All reagents need to be brought to room temperature prior the assay.
       Wash Solution
        Dilute 50 ml of Wash Solution concentrate with 450 ml of deionized (distilled) water. The diluted Wash Solution is stable for one month if stored at 2-8°C.
      Dilution Buffer
      Dilute 25 ml of Dilution Buffer concentrate with 75 ml of deionized (distilled) water. The
diluted Dilution Buffer is stable for one month if stored at 2-8°C.
      SP-D Calibrators
Reconstitute each Calibrator with 0.25 ml of distilled water and mix gently (manually rotate the vial). Allow it to sit at least 5 minutes. It is necessary for ensure complete reconstitution.The concentration of the SP-D in Calibrators are follows:

Calibrator         Distilled water       Resulting concentration
Calibrator 100       250 µl                   100 ng/ml
Calibrator 50         250 µl                     50 ng/ml
Calibrator 25         250 µl                     25 ng/ml
Calibrator 12.5      250 µl                    12.5 ng/ml
Calibrator 6.25      250 µl                    6.25 ng/ml
Calibrator 3.13      250 µl                    3.13 ng/ml
Calibrator 1.56      250 µl                    1.56 ng/ml

Reconstituted Calibrators are ready to use, do not dilute them. Reconstituted calibrator solutions are stable until the expiration date (see label on the box) if stored at -20°C.

Quality Controls
Reconstitute each Quality Controls with 0.25 ml of distilled water (refer to the Certificate of Analysis for actual Quality Controls values). Reconstituted Quality Controls are ready to use, do not dilute them. Reconstituted Quality Controls are stable until the expiration date (see label on the box) if stored at 2-8°C.

Conjugate Solution
Add 0.05 ml of concentrated of Conjugate Solution to 11 ml of Dilution Buffer and shake gently.
This solution should always be prepared fresh. Store stock Conjugate Solutoin only. Stock Conjugate Solution is stable until the expiration date (see label on the box) if stored at 2-8°C.

Samples
Dilute serum or plasma samples 1:11 with Dilution Buffer just prior to use in the assay, e.g. 25 µl of sample + 250 µl of Dilution Buffer. Recommended dilution for BAL samples is 1:40, e.g. 10 µl of BAL sample + 390 µl of Dilution Buffer. Serum and plasma samples are stable for 1 year if stored at –20°C and for 2 years if stored at –70°C. BAL samples are stable for 1 year if stored at -70°C.

Note:
If you do not use the whole plate, return unused strips in the provided aluminium bag and seal the bag carefully. Keep the unused strips at 2-8°C, protected from the moisture.
Results exceeding Surfactant Protein D level of 100 ng/ml should be repeated with more diluted samples. Dilution factors need to be also taken into consideration in calculating the Surfactant Protein D concentration.