Direct, sensitive and specific elisa of plasma estriol in pregnancy

IMPROVEMENTOF MICROTITRATION PLATE ENZYME IMMUNOASSAYSFOR 'SECOND ANTIBODY TECHNIDUE'; Meyer, H.H.D.; Guven, 8.

~ Milchwirtachaft,

Techn.

Institut Universitat

Microtitration plate enzyme nations. Troubling problems

fur Physiologie, Siidd.Versuchsu. MOnchen, 8050 Freising-Weihenstephan;

immunoassay5

are

STEROID

large

(EIA) sre variations

rarely due

BY

A

Forachungsanstelt fur WEST-GERMANY (FRGj

up to now for varying'coetability

used to

DETERMINATION

steroid determiof the wells

resulting in different amounts of directly bound, hormone specific antibody and 'head tail phenomena' within the test. Therefore 5 new incubation technique wsa developed. Experimental: Serum from a sheep, that had been immunized with rabbit IgG, was stirred overnight with rebbit-IgG-agarose, The gel was transferred to 5 column, washed and then eluted with 2 M NaSCN. 0.5 @g affinity isolated sheep IgG were coated per well, the plates were washed and enzyme labeled steroid plus standard or sample were edded. Immunreactions started after dispensing the steroid specific rabbit antiserum. After incubation at O°C overnight, the plates were washed and evaluated after enzyme reaction. Results: The second antibody technique shows several advanteges: Variebilitiea of colour readings are reduced (CV-3.2% instead ll.YX in the former technique), about 5-10 times less antibody is required, and coated plates can be stored at O°C or -2O'C without loosing precision. Teats were developed for progesterone, eatradiol, trenbolone and 19-norteatoaterone. Moat of these teats are more sensitive than the corresponding RIA. Discussion: The sheep antibody is coated in a large excess and therefore the hormone specific antibody is bound completely, resulting in a low variability. The teat for the anabolic agent trenbolone is already successfully used for monitoring the application of this steroid in lifestock oroduction.

_

140 DIRECT, SENSITIM: AI’D SPECIFIC ELIs4 C.F PLA&vlAES’lRlU Magrini O., Preti MS., Cristiani P. of Reproductive Medicine, S.Orsola General via Massarenti 13, 40138 Bologna,Italy.

t

IN FREQWCf

and Flsrnigni C. Hospital, University

of Bologna,

A cunpetitive,sensitive and rapid enzyme-linked imnmoadsorbent assay (ELIS4) was developed for the determination of unconjugated estriol in unextracted plasma or serun. A highly specific antisertxn (anti-6oxoestriol-6-C.h4.0.-BS4) was used in this method. The cross-reactions of the antiserum for estrioi-3-sulfate and estriol-16sr-glucoronide were<2% and
-141

DIRECT , SENSITIVE AND SPECIFIC ELISA OF PLASMA PROGESTERONE O., Lodi S., Preti M.S., Cristiani P. and Flamigni C. Department of Reproductive S. Orsola General Hospital, University of Bologna,via Massarenti 13,40138 Bologna, Italy. Magrini

Medicine

1

A competitive and sensitive enzyme-linked immunoadsorbent assay (ELlSA) was developed for the de antiserum termination of progesterone in unextracted plasma or serum. A specific and highly sensitive (anti-progesterone-3-C.M.O.-BSA) was used in this method. The high sensitivity of the method ( 2 pg/tube) permitted the assay on 10 JII of plasme samples. The reduction of plasma proteins hip ding capacity was obtained by adding 8-Anilinonaphthalene-I-sulfonic acid ammoniun salt (ANSA). Progesterone-3-C.M.O. was coupled with horseradish peroxidase (HRP) by the mixed anhydride method. The free-bound separation was carried out by adsorbing purified IgG of antiserum on polystyrene balls. The enzyme activity was measured, after incubation, by a calorimetric reactlon using OPD.2HCI and H202 as substrate. In order to compare ELISA to RIA progesterone estimations, a total of 28 ‘plasma samples of women during various phases of cycle were assayed. RIA was performed by extrac tion method, A good correlation was found between the results obtained by ELISA and RIA: r10.992, p
50s

I