| Enzyme Amplification, A Method Applied to Provide An ELISA
for Studying Anti-Hib-Ps Antibodies in Children |
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Kyung Hyo Kim1, Moon H. Nahm2 |
1Department of Pediatrics, College of Medicine, EwhaWoman`s University, Seoul, Kore
2Departments of Pathology and Medicine, Washington University School of Medicine, USA |
| 소아의 항Hib-PS항체 측정을 위한 효소 증폭 면역법의 적용 |
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김경효1, Moon H. Nahm2 |
1이화여자대학교 의과대학 소아과학교실
2Departments of Pathology and Medicine, Washington University School of Medicine, USA |
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| Abstract |
Purpose
: Enzyme immunoassay (EIA) is now a widely used technique. We have
described the application of enzyme amplification (sensitive ELISA) in the field of
immunoassays of pediatric population. There are two issues with the sensitive ELISA.
First is that one can minimize the serum volume, an important concern for pediatricians. The second is the problem of background signal. We demonstrate that it is possible to develop EIAs ofhigh sensitivity and detectability with using very smallvolume of infant's sera immunized with Hib-PRP vaccine.
Methods
: Monoclonal Abs HG11, HP6016, HK2, and KL1 specific for human IgG1,
IgG2, Cκ, and Cλ were used. The mAb OAK-1 specific fora subfamily ofVκIL
chains(VκIa),the mAbs KB13 and B12 specific forhuman VκIIand VκIIIL chains
were also used respectively.
Adults were immunized with Hib-CRM vaccine. Immune serum was obtained 4 to 8
wk after immunization. Twenty infants received Hib-CRM vaccine at 2, 4, and 6 month
of age and blood samples were obtained at 7 month old. The amount of anti-Hib-PS
Ab expressing aVκ subgroup or Vλ was determined by sandwich type immunoassays
using conventional substrate. The amount of the enzyme immobilized to the well was
determined with para-nitrophenyl phosphate substrate. A standard ELISA was
performed but different substrate (lyophilized NADPH) and amplifier (alcohol dehydrogenase and diaphorase) were used to develop color in final step for enzyme
amplification method.
Results
: We get the dose-response curves obtained using the conventional and
amplified detection methods in the anti-PRP Ab assay. The sensitivities ofthe two
assay methods were compared. We can increase the sensitivities four to sixteen folds
and minimize the infant's sera volume to perform varing anti-PRP antibody assays. To
obtain the advantages of increased sensitivity, any background is minimized by using
noncontaminated reagents.
Conclusion
: It is possible to develop EIAs of high sensitivity and detectability
with using very small volume of infant's sera with using enzyme amplification system
(sensitive ELISA). |
| Key Words:
ELISA, Enzyme amplification |
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