Susceptibility of immunoassays to interference from human anti-animal antibodies: How common a problem ?

1. Susceptibility of immunoassays to interference from human anti-animal antibodies: How common a problem? Roger L. Bertholf, Ph.D. Associate Professor of Pathology University of Florida Health Science Center/Jax

2. Human anti-animal antibodies • First described in 1971 by Ammann and Hong • Potential to cause positive or negative interferences • Anti-isotype or anti-idiotype • Anti-mouse antibodies are most common • Incidence is not certain

3. Substrate E E E E E E 2nd antibody Specimen S P Two-site “sandwich” enzyme immunoassay

4. Substrate E E E E E 2nd antibody S P HAMA “bridging” interference

5. E E E E HAMA blocking interference

6. Labeled antigen Specimen Wash Competitive immunoassay

7. HAMA blocking interference

8. Case Report The laboratory director was consulted by a gynecologist about discrepant CA-125 results on a patient being monitored for recurrence of a surgically removed ovarian tumor. CA-125 results from a referral laboratory had been consistently high and increasing, while results from the hospital laboratory were within normal limits.

9. CA-125 • A large glycoprotein expressed by tissues originating from the müllerian ducts • Mab OC 125 was isolated by Bast and colleagues in 1981, using cells from a serous papillary cystadenocarcinoma • Useful for detecting and monitoring ovarian tumors • Also elevated in endometrial, pancreatic, breast, colorectal, lung and GI carcinomas

10. Clinical utility of CA-125 • Lacks sensitivity and specificity as a screening test for ovarian cancer • Normal in 50% of stage I ovarian cancers • PV+ 5% • Very sensitive indicator of recurrence following surgical resection • Failure to return to normal after chemotherapy is indicative of drug-resistant disease

11. Referral Lab SHJ

12. AbbottRoche

13. Follow-up studies • HAMA quantitation (IRMA)* • Result = 196 ng/mL (0 – 188) • CA-125 with heterophilic blocking reagent* • Without blocking reagent: 211.5 U/mL • With blocking reagent: 11.0 U/mL *Scantibodies, Inc., Santee, CA

14. Sources of foreign IgG exposure • Imaging reagents • Pharmaceuticals • Vaccines • Abzymes (proposed)

15. Incidence of HAMA • Clinical trials • 0-100% reported • Population studies • 72 of 10,000 blood donors (1990 abs.) • 92 of 1,008 blood donors (1986, indir.) • 51 of 67 blood donors (1991) • 8 of 10 infants (1991)

16. Conclusions • HAMA can cause clinically significant elevations in CA-125 assays • Different immunoassays may not respond equally to the presence of HAMA • The population frequency of HAMA is not known, but is likely to increase • Interference may occur at modest concentrations of HAMA

17. “Beware of false knowledge; it is more dangerous than ignorance.” - George Bernard Shaw (1856–1950) “Something is better than nothing, unless ‘something’ is wrong, in which case nothing is better than something.” - John Savory