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ELISA protocol (human IgG4 antibody)


FR3 and Dr. Gary Weil are providing antigens and a suggested protocol as a service to the filariasis research community. FR3 will not ship antigens until they receive a signed Conditions of Use document for the Bm14 antigen. This is not a kit!  Some work will be required by each lab to standardize the test with their own conditions, and experience with ELISA is needed to get good, consistent results.

Antibody monitoring: The Bm14 antibody assay tests for active infection or heavy exposure to filarial parasites. Early antibody diagnostic tests for LF based on detection of IgG antibodies to native antigens were plagued by poor specificity. Our group achieved greatly improved specificity by testing for IgG4 antibodies to a recombinant filarial antigen, Bm14 (ORF 459 bp). 1 This antigen is similar to Bm SXP-1 reported by Piessens’s group.2 Numerous studies have shown that the Bm14 antibody test is sensitive for infection with (or heavy exposure to) B. malayi and W. bancrofti (generally positive in over 90% of MF carriers).3 A recent blinded multicenter study confirmed this finding.4 Primates produce antibodies to Bm14 a few weeks after they are infected with Brugia (i.e., during the pre-patent period). Humans with pre-patent infections also have antibodies to Bm14; a prospective study showed that antibody to Bm14 was a significant risk factor for incidence of microfilaremia over the next year.5 While a positive antibody test does not prove current infection, this test is specific for infection or heavy exposure to filarial parasites (i.e. no false-positive tests with sera from people with or without other nematode infections who have not been exposed to filarial parasites). Prior studies have shown that antibody prevalence rates are much higher than MF and antigen prevalence rates in low-prevalence settings (pre-MDA Egyptian villages)5, but these three measures of filariasis activity varied in parallel in untreated populations. Antibody rates also tend to be much higher in young children in areas with low-level LF transmission than antigen or MF rates. For this reason, MF and filarial antigen tests are not as useful as antibody testing for use in sentinel children in assessing changes in LF transmission following MDA; preliminary studies (Ramzy et al and Weil et al, unpublished data) have shown that antibody rates in young children fall fairly rapidly in the years following initiation of effective MDA programs. On the other hand, recent studies have also shown that IgG4 antibodies to Bm14 decrease very slowly following DEC/Alb treatment even in subjects who clear MF and antigenemia. 6.

Please contact Gary Weil ( for questions regarding the Bm14 antibody assay.


  1. Chandrashekar R, Curtis KC, Ramzy RM, Liftis F, Li B-W, Weil GJ, 1994. Molecular cloning of Brugia malayi antigens for diagnosis of lymphatic filariasis. Mol Biochem Parasitol 64: 261-274.
  2. Dissanayake S, Xu M, Piessens W, 1992. A cloned antigen for serological diagnosis of Wuchereria bancrofti microfilaremia with daytime blood samples. Mol Biochem Parasitol 56: 269-277.
  3. Ramzy R, Helmy H, Faris R, Gad A, Chandrashekar R, Weil G, 1995. Evaluation of a recombinant antigen-based antibody assay for diagnosis of bancroftian filariasis in Egypt. Ann Trop Med Parasitol 89: 443-446.
  4. Lammie P, Weil G, Rahmah N, Kaliraj P, Steel C, Goodman D, Lakshmikanthan V, Ottesen E, 2004. Recombinant antigen based assays for the diagnosis and surveillance of lymphatic filariasis  - a multicenter trial. Filaria Journal 3: 9.
  5. Weil GJ, Ramzy RM, El Setouhy M, Kandil AM, Ahmed ES, Faris R, 1999. A longitudinal study of Bancroftian filariasis in the Nile Delta of Egypt: baseline data and one-year follow-up [In Process Citation]. Am J Trop Med Hyg 61: 53-8.
  6. Helmy H, GJ W, As E, ES A, M E-S, RMR R, 2005. Bancroftian filariasis: Effect of repeated treatment with diethylcarbamazine and albendazole on microfilaremia, antigenemia, and anti-filarial antibodies. Trans Roy Soc Trop Med in press.


Suggested protocol for the Bm14 IgG4 Antibody Assay


  1. Sensitize a 96 well plate with 100ul Bm14-HIS per well (2ug Bm14-HIS per ml in carbonate buffer, pH 9.6) overnight 37C.  Sensitize another plate with 2 ug/ml control Cat-HIS antigen in carbonate buffer. 
  2.  Next morning shake out the antigen solution, wash 3 times with PBS/Tween, and block by adding 200 ul per well PBS/T/FCS (PBS/Tween with 5% v/v fetal calf serum) 30 minutes at 37C.
  3.  Wash plate 3 times with PBS/Tween.
  4.  Dilute sera 1:100 in PBS/T/FCS (5 ul serum plus 495 ul diluent). 
  • For serum sets expected to have many positives: Test sera in duplicate wells on each of the plates (100ul of 1:100 dilution per well), incubate 2 hours 37C. Include controls; 2 wells each of a positive filarial antibody serum pool and a nonendemic normal human serum pool.  Each lab must produce and test its own positive and negative control samples. We dilute our positive pool so that it produces a net O.D. between 2.0 and 3.0 (i.e., not “over” or starring out the reader).
  • For serum sets expected to have few positives: Test one well per serum. Retest positives in another run with duplicate wells for conformation.
  • Wash plates 3 times with PBS/Tween.
  • Conjugate: Add 100ul peroxidase labeled anti-human IgG4. Working dilution must be worked out by your lab.
  • Wash plate 3 times with PBS/Tween.
  • Substrate:  Add 100 ul OPD substrate solution per well, and incubate in the dark, 10min, room temperature.  (Other substrates such as premixed ABTS or TMB can be used, but we have not used these extensively). 
  • Stop the OPD substrate reaction by adding 50ul of 4 M sulfuric acid to each well.  (Be careful with acid !  Add acid to water for dilution).
  • Scoring results:  Read OD 490 and calculate mean OD values for each serum tested.  (When duplicate values do not agree regarding positivity, the serum should be retested).  (Note that it is desirable to use 490 with a second reference wavelength over 600 nm if possible to correct for imperfections in ELISA plates). (We keep aside wells G11 and G12 as blanks and blank the reader with water in those wells). Subtract mean OD values on the Cat-HIS plate from those on the Bm14-HIS plate to get the mean net OD.  The cutoff net OD value is the mean plus 3 SD of the net OD values obtained with the panel of negative sera.  Cutoff values will vary from lab to lab, but they should be in the range of 0.100 to 0.200.  Note that most sera will give very low OD values with the Cat-HIS control antigen.  However, we like to test the sera in parallel to catch the unusual serum that reacts with Cat-HIS in the IgG4 assay.  You may find that this is not necessary. Your positive control pool is your proof that the assay is working properly.
  • Note:  Before you start testing your sera:  It is necessary to start with a technical run with 10 to 20 of your own nonendemic (not exposed to filariasis) sera and a few of your own positive sera to make sure the reagents are working and to define the background cutoff for the assay in your laboratory. We use the mean plus 3 SD of the net OD obtained with the panel of negative sera to define the cutoff for a positive test. This tends to be approx 0.150 in our lab conditions. Control serum samples generally look water clear in the ELISA and wells in the CAT-his control antigen plate are also usually water clear. If a serum produces an O.D. value with the control antigen that is as high or higher than that produced on the Bm14 plate, the result is indeterminate/invalid. 

    Carbonate buffer (0.06M, pH 9.6)(for sensitizing plates with antigen):

    • Buffer A: 1.0M Sodium Bicarbonate  (NaHCO3) 8.4g in 100 ml dH2O.
    • Buffer B: 1.0M Sodium Carbonate  (Na2CO3) 10.6g in 100 ml dH2O.
    • Combine 22.65 ml Buffer A, with 9.1 ml Buffer B, and dilute to 500 ml with dH2O.


    PBS Stock Solution to make PBS/Tween Wash 

    • 54.8g    Sodium Phosphate Dibasic (Na2HPO4) into 772 ml dH2O.
    • 15.75g Sodium Phosphate Monobasic (NaH2PO4) into 228 ml dH2O.
    • Dilute to 2000ml with d dH2O


    PBS/Tween Wash (Tween concentration is 0.05%)

    • 240ml    PBS Stock Solution
    • 51g Sodium Chloride NaCl
    • 3.0 ml     Tween 20 (Sigma)
    • Dilute to 6000 ml with dH2O.

    PBS/T/FCS (Blocking solution and diluent for sera)

    • PBS/Tween with 5% fetal calf serum (heat inactivated serum, 56 C for ½ hr)

    Citrate Buffer (for OPD substrate):   

    • Buffer A.  9.6g Citric Acid, 500ml dH2O.
    • Buffer B.  26.87g Dibasic Sodium Phosphate (Na2HPO4) in 500 ml dH2O.
    • Add 486 ml of A. to 500 ml B. Adjust pH to 5.0 

    OPD substrate solution:

    • 4mg OPD (O-phenylenediamine, Sigma Chem. Co., St. Louis, MO, USA) in 10ml Citrate buffer, 2 ul of 30% H2O2.  OPD is poorly soluble and must be added at least 20 minutes before you plan to use the substrate.  Add H2O2 at the last minute.  (Substrate must be made up daily. Keep substrate in the dark until you use it.  Plates with substrate must be incubated in the dark). 

    ELISA PLATES:  We have found that polyvinyl plates work best in this assay.  These can be flat or round bottomed (for example, Vinyl U bottom plates, Part No. 2401,ThermoLab Systems, 8 East Forge Parkway, Franklin, MA 02038. Tel 800 522 7763). Immunlon 4 plates do not work well.