Assays for Complement Proteins Encoded in the Class III Region of Human MHC

Lilian Varga1, George Füst1

1 Semmelweis Medical University, Budapest
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 13.7
DOI:  10.1002/0471142735.im1307s67
Online Posting Date:  July, 2005
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Abstract

Investigation of the complement proteins encoded in the class III region of the human MHC isclinically significant. A low level of components that make up the classical pathway is indicative of an underlying in vivo complement activation or primer complement deficiency. Hemolytic tests of C4 and C2 described are the only methods that allow quantitification of the functional complement proteins. The application of these sensitive techniques is unavoidable if inactive complement proteins are present. The complex hemolytic titration process protocol described in detail makes possible the use of this test. Assays to determine complement components belonging to the MHC III region were completed by a basic phenotyping test of the standard factor B alleles. This simple technique is a very useful firstā€step assay for clinical laboratories engaged in genetic studies.

Keywords: functional C2; functional C4; factor B phenotyping; MHC III; hemolytic assay; HVAGE

     
 
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Table of Contents

  • Basic Protocol 1: C4 Hemolytic Assay
  • Basic Protocol 2: C2 Hemolytic Assay
  • Support Protocol 1: Preparation of EAC1
  • Support Protocol 2: Preparation of EAC14
  • Support Protocol 3: Preparation of Functionally Pure Guinea Pig C1
  • Support Protocol 4: Preparation of C2
  • Support Protocol 5: Determination of Tmax for EAC14
  • Basic Protocol 3: Phenotyping Human Factor B
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: C4 Hemolytic Assay

  Materials
  • Test serum sample (see )
  • Control serum (see recipe)
  • LIB buffer (see recipe), ice cold
  • 1 × 108 cell/ml EAC1 suspension (see protocol 3)
  • C2 (see protocol 6)
  • C‐EDTA (see recipe)
  • 0.04 M EDTA (see recipe for 0.1 M EDTA)
  • 0.15 M NaCl, ice‐cold
  • Disposable 16 × 100–mm borosilicate glass culture tubes
  • Repeating pipettor
  • 30° and 37°C incubator or water bath
  • Refrigerated centrifuge with cariers for 16 × 100–mm and 50‐ml tubes
  • Spectrophotometer
  • Linear graph paper or computer software (e.g., Excel, GraphPad Prism, or equivalent)

Basic Protocol 2: C2 Hemolytic Assay

  Materials
  • Test serum sample (See )
  • Control serum (see recipe)
  • LIB buffer (see recipe), ice cold
  • 1 × 108 cell/ml EAC14 suspension with predetermined T max (see Support Protocols protocol 42 and protocol 75)
  • C‐EDTA (see recipe)
  • 0.04 M EDTA (see recipe for 0.1 M)
  • 0.15 M NaCl, ice‐cold
  • Disposable 16 × 100–mm borosilicate glass culture tubes
  • Repeating pipettor
  • 30° and 37°C water baths or incubators
  • Centrifuge with carriers for 16 × 100–mm borosilicate glass culture tubes
  • Spectrophotometer

Support Protocol 1: Preparation of EAC1

  Materials
  • Sheep whole blood in Alsever's solution (e.g., from Colorado Serum)
  • GVB++ buffer (see recipe)
  • Hemolysin (see recipe)
  • LIB buffer (see recipe)
  • C1 (see protocol 5)
  • Disposable 16 × 100–mm borosilicate glass culture tubes
  • 100‐ml glass flasks
  • Centrifuge with carriers for 16 × 100–mm and 50‐ml tubes
  • Spectrophotometer
NOTE: All reagents, cells, and sera are to be kept on ice throughout this protocol unless otherwise noted.

Support Protocol 2: Preparation of EAC14

  Materials
  • EA cells (see protocol 3, steps to )
  • LIB buffer (see recipe), ice cold
  • GVB++ buffer (see recipe)
  • C1 (see protocol 5)
  • Fresh normal human serum (NHS)
  • GVBE buffer (see recipe)
  • Disposable 16 × 100–mm borosilicate glass culture tubes
  • 100‐ml glass flasks
  • Centrifuge with carriers for 16 × 100–mm borosilicate glass culture tubes
  • 37°C water bath
NOTE: All reagents, cells, and sera are to be kept on ice throughout this protocol unless otherwise noted.

Support Protocol 3: Preparation of Functionally Pure Guinea Pig C1

  Materials
  • Dimethylformamide (N,N‐dimethylformamide)
  • Fresh (or frozen at −70°C) guinea pig serum
  • 1 M CaCl 2 (see recipe)
  • 0.3 M NaCl
  • 100‐ml centrifuge bottles
  • Centrifuge with carriers for 100‐ml centrifuge bottles
NOTE: All reagents, cells, and sera are to be kept on ice throughout this protocol unless otherwise noted.

Support Protocol 4: Preparation of C2

  Materials
  • 10 ml fresh guinea pig serum or byproduct of C1 preparation (see protocol 5)
  • Saturated ammonium sulfate (saturated (NH 4) 2SO 4)
  • Diluted (NH 4) 2SO 4 solution (270 ml saturated (NH 4) 2SO 4 plus 100 ml water)
  • VBS++ (see recipe)
  • Iodine solution (see recipe)
  • Glucose
  • Dialysis sack (average flat width 9 mm)
NOTE: All reagents, cells, and sera are to be kept on ice throughout this protocol unless otherwise noted.

Support Protocol 5: Determination of Tmax for EAC14

  Materials
  • Normal pooled serum (NPS)
  • 1 × 108 cell/ml EAC14 suspension (see Support Protocols protocol 53 and protocol 64)
  • C‐EDTA (see recipe)
  • Additional reagents and equipment for serum dilutions and termination of C2 hemolytic assay (see protocol 2)

Basic Protocol 3: Phenotyping Human Factor B

  Materials
  • Agarose gel (see recipe)
  • Electrode buffer (see recipe)
  • Serum or EDTA‐plasma samples (fresh or frozen at −70°C)
  • Control sera (BF*F, BF*S, and FS allotypes)
  • Hemoglobin A (Sigma)
  • Monospecific anti‐BF antiserum (DiaSorin)
  • 0.15 M NaCl
  • 0.2% Coomassie brilliant blue R‐250 (Sigma)
  • Destaining solution (see recipe)
  • 265 × 125–mm GEL‐FIX supporting film for agarose (SERVA Electrophoresis)
  • Leveling table
  • Humid chamber: plastic box with cover and two layers of Whatman no. 3 paper layering the bottom, humidified by water
  • Plastic film pattern for making 25 5 × 1–mm sample slits
  • Scalpel
  • Whatman no. 3 filter paper
  • Glass rod
  • Additional reagents and equipment for gel electrophoresis (unit 8.4)
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Figures

Videos

Literature Cited

Literature Cited
   Alper, C.A. and Propp, R.P. 1968. Genetic polymorphism of the third component of human complement (C3). J. Clin. Invest. 47:2181‐2191.
   Alper, C.A. and Johnson, A.M. 1969. Immunofixation electrophoresis: A technique for the study of protein polymorphism. Vox Sang. 17:445‐452.
   Alper, C.A., Boenisch, T., and Watson, L. 1972. Genetic polymorphism in human glycine‐rich beta‐glycoprotein. J. Exp. Med. 135:68‐80.
   Alper, C.A., Awdeh, Z.L., and Yunis, E.J. 1986. Complotypes, extended haplotypes, male segregation distortion, and disease markers. Hum. Immunol. 15:366‐373.
   Davidson, I.A. and Dyer, P.A 1984. Allotypes of properdin factor B (Bf) and lymphocytotoxic antibody production. Complement 1:52‐57.
   Füst, G., Czink, E., Minh, D., Miszlay, Z., Varga, L., and Hollán, S.R. 1985. Depressed classical complement pathway activities in chronic lymphocytic leukaemia. Clin. Exp. Immunol. 60:489‐495.
   Gaither, T.A., Alling, D.W., and Frank, M.M. 1974. A new one‐step method for the functional assay of the fourth component (C4) of human and guinea pig complement. J. Immunol. 113:574‐583.
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   Geserick, G. and Patzelt, D. 1988. Factor B (BF) subtyping by isoelectric focusing: Methods, nomenclatures, genetics and forensic application. Electrophoresis 9:418‐421.
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   Siemens, I., Bender, K., Geserick, G., Mauff, G., and Pulverer, G. 1989. The Bf F subtypes are detectable in the Ba fragment of factor B. Forensic Sci. Int. 42:279‐286.
   Suzuki, K., Haramuto, T., Ito, S., and Matsumoto, H. 1987. Subtyping of factor B by agarose electrophoresis. Electrophoresis 8:481‐485.
   Tamura, N. and Nelson, R.A. 1968. The purification and reactivity of the first component of complement from guinea pigs, human and canine sera. J. Immunol. 101:1333‐1345.
   Teisberg, P. 1970. High‐voltage gel electrophoresis in the study of C3 polymorphism. Vox Sang. 19:47‐56.
   Teng, Y.S. and Tan, S.G. 1982. Subtyping of properdinfactor B (Bf) by isoelectrofocusing. Hum. Hered. 32:362‐366.
   Varga, L., Felicitas, C., Füst, G., Pálóczi, K., Szegedi, G., Loos, M., and Hollán, S.R. 1988. Patients with Cll and Hypokoplementaemia have impaired serum bactericidal activity against the Salmonella Minnesota Re mutant. Complement 5:40‐45.
   Varga, L., Czink, E., Miszlai, Zs., Pálóczi, K., Bányai, A., Szegedi. G., and Füst, G. 1995a. Low activity of the classical complement pathway predicts short survival of patients with chronic lymphocytic leukaemia. Clin. Exp. Immunol. 99:112‐116.
   Varga, L., Poros, A., Puskás, É., Pánya, A., Kramer, J., Gyódi, É., and Füst, G. 1995b. Clinical significance of longitudinal complement measurement in recipients of bone marrow transplant. Bone Marrow Transplant. 15:509‐514.
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